The isolation of leaf protoplasts is a stress-inducing procedure that generates reactive oxygen species (ROS). We found that a cellulase-pectinase enzyme solution used for isolating protoplasts contained peroxidase (POX), catalase (CAT), and superoxide dismutase (SOD) activity, and effectively scavenged externally added hydrogen peroxides. During the isolation of Brassica napus leaf protoplasts, the protoplasts accumulated 1.5 times more intracellular H2O2 than normal leaf cells, but the H2O2 released into the medium increased only negligibly during culture. H2DCF-DA and DAF2-DA were used as probes for the intracellular localization of H2O2 and NO with a confocal laser scanning system. H2O2 and NO had different intracellular localizations in freshly prepared B. napus leaf protoplasts. These results indicate that intracellular ROS are generated by the stresses of protoplast isolation and will induce the apoptosis-like cell death of the cultured protoplasts.
Previously, we reported on an inducible viral vector system for foreign protein production in cultured plant cells, in which the transcription of the recombinant viral vector RNA encoding a foreign protein is controlled by an estradiol-inducible promoter. In this study, we used the inducible virus vector system to test the efficiency of a modified tomato mosaic virus (ToMV) encoding the movement protein. The virus inducibly produced a foreign jellyfish green fluorescent protein encoded in the virus in transgenic tobacco BY-2 suspension-cultured cells as efficiently as modified ToMV without the movement protein. We then produced transgenic BY-2 cell lines ER–ToMV–MP–DHFR and ER–ToMV–MP–GUS, which encoded modified ToMV with Escherichia coli dihydrofolate reductase (DHFR) and β-glucuronidase (GUS), respectively. After estradiol was added to the cell culture, DHFR and GUS activity was detected in the ER–ToMV–MP–DHFR and ER–ToMV–MP–GUS cells, respectively. In contrast, no DHFR or GUS activity was detected in untreated transgenic lines. Three days after induction, DHFR accumulation accounted for up to 15% of the total soluble proteins extracted from the cells, indicating that an inducible viral vector is an effective option for efficiently producing active enzymes in cultured plant cells.
In Nicotiana suaveolens × N. tabacum hybrid cells incubated with high levels (0.8, 4.0 or 20 μM) of BAP at 28°C, the percentage of dead cells and extent of nuclear fragmentation were suppressed and some cultured cells remained green compared with those incubated with a standard level (0.04 μM) of BAP. Though DNA ladders were detected in all hybrid cells incubated at 28°C, positive signals for TUNEL assays were detected less frequently in hybrid cells incubated with high levels of BAP than in those incubated with a standard level of BAP. The localized accumulation of formazan, indicating production of reactive oxygen species, in hybrid cells with high levels of BAP was less than that in hybrid cells with a standard level of BAP. These results suggest that high levels of BAP suppress programmed cell death in hybrid lethality.
Specifically accessible chromatin sites, detected as DNase I hypersensitive sites (DNase I HSs), have been found at promoters of most genes in yeast, human as well as in Arabidopsis chromatin. The DNase I HSs are constitutively present at promoters of most genes both at inactive and activated states. On the other hand, appearance and disappearance of DNase I HSs have been observed at several developmentally regulated genes in different tissues of animals. Patterns of appearance and disappearance of DNase I HSs were correlated with transcriptional competency (potential of expression) of the associated genes, suggesting that DNase I HSs are absent from the promoters of transcriptionally incompetent (unexpressed nor inducible) genes. To test this hypothesis in plants, we analyzed DNase I hypersensitivity of several well-characterized genes showing distinct patterns of tissue-specific expression in Arabidopsis leaves, roots and suspension cells. Distinct DNase I HSs were present at transcriptionally competent gene promoters, but absent from incompetent gene promoters. The absence of DNase I HS suggests that nucleosomes are randomly positioned on incompetent promoters, and may contribute to the transcriptional incompetency by blocking the binding of activators in tissues where expression of these genes is unnecessary.
In higher plants, cytochrome P450 (P450, CYP) monooxygenases play important roles in the biosynthesis and metabolism of endogenous compounds and/or in the oxidative reactions of xenobiotics, such as herbicides, insecticides, and fungicides. In this study, the novel P450 cDNAs encoding CYP71A12 was isolated from wild-type Arabidopsis thaliana ecotype Ws using an RT-PCR strategy. Northern blot analysis revealed that cyp71A12 is expressed specifically in roots but not in flowers, leaves, or stems. To determine the function of CYP71A12, the coding sequences of the corresponding cDNAs were expressed in Saccharomyces cerevisiae AH22 cells. Microsomal fractions expressing CYP71A12 metabolized the herbicide pyrazoxyfen. Liquid chromatography mass spectrometry (LC/MS) analysis indicated that the metabolite of pyrazoxyfen might be N-demethylated at the pyrazole ring and 5-hydroxylated at the dichlorobenzene ring. This is the first report that a pyrazole herbicide is metabolized by a plant P450 species.
Two genes (fad3a and fad3c) encode for endoplasmic delta-15 linoleate desaturase, which is responsible for the desaturation of linoleic acid (C18:2) into linolenic acid (C18:3) in canola (Brassica napus). The canola mutant line DMS100 carries a G-to-A base substitution at the 5′ splice site, located at +1 G, of fad3c intron 6 and contains reduced C18:3 content in oil seeds. Reverse transcription (RT) PCR analysis was used to amplify part of the fad3c transcript spanning intron 5, exon 6 and intron 6 using the primers specific to the exon 5 and 7 to investigate whether the mutation caused any abnormal splicing. The RT-PCR amplified a fragment of larger size in the fad3c mutant than in wild-type. Sequencing of the RT-PCR fragments revealed that the mutation activated an impaired splicing which retained the entire intron 6 in the mature fad3c transcript of the mutant. The impaired splicing could result in early termination of translation and synthesis of a shorter fad3c polypeptide because the intron 6 contained stop codons in all three possible reading frames. The incomplete translation could produce an inactive enzyme which blocked the desaturation of linoleic acid (C18:2) to linolenic acid (C18:3), resulting in the decrease of C18:3 accumulation in canola seeds. This is consistent with the observation that the fad3c mutant has significantly lower C18:3 content (<3%) than the wild type (~7%).
We isolated an SNF1-related kinase 2 homolog (SlSnRK2C) from tomato (Solanum lycopersicum L. cv. Micro-Tom) which has significant similarlity to AtSRK2C (72% amino acid identity). We profiled expression levels and biochemical properties of SlSnRK2C. In contrast to AtSRK2C which phosphorylates histone H1 and is mainly expressed in root chip, SlSnRK2C phosphorylates myelin basic protein but not histone H1 and SlSnRK2C mRNA is expressed in leaves, flowers and fruits but not in roots. Immunoblot by anti-SlSnRK2C-COOH terminus specific antiserum shows that SlSnRK2C is expressed remarkably in developing fruits, and weakly in flowers, mature green fruits and shoots, but marginally in mature red fruits or roots. SlSnRK2C is stimulated strongly by chilling and NaCl stress. These observations suggests that SlSnRK2C is possibly involved in fruit development and stress signaling in response to salt and chiling stress.
Ralstonia solanacearum is a causal agent of bacterial wilt, and the bacterium normally infects through wounds in roots. Since it is not possible to equally and simultaneously inoculate the host plants with bacteria via root inoculation, it has proven difficult to elucidate the molecular events in plants infected with R. solanacearum. To improve the efficiency of inoculation, we inoculated tobacco plants with R. solanacearum by leaf-infiltration. Differential display was carried out to isolate fragments of genes that are regulated in tobacco by virulent strains of R. solanacearum OE1-1 and by the avirulent mutant of R. solanacearum, 31b, which is mutated in the type III secreted PopA protein by transposon insertion. Nineteen R. solanacearum-responsive genes (RsRGs) were successfully isolated using equalized cDNA libraries constructed with water-, R. solanacearum OE1-1- and 31b-infiltrated tobacco leaves. From Northern blot analysis, RsRGs were divided into 3 groups; 1) responsive to both virulent and avirulent bacteria (8 RsRGs), 2) responsive to avirulent bacteria (3 RsRGs), and 3) responsive to virulent bacteria (3 RsRGs). These results suggest that the cDNA pool and method presented in this study provide a valuable resource for functional genomic analysis of R. solanacearum-plant interactions.
Identification and characterization of germplasm is an important link between the conservation and utilization of plant genetic resources. The present investigation was undertaken for identification and genetic variation within eight high yielding varieties of ginger through RAPD markers. A total of 55 distinct DNA fragments ranging from 0.5–2.4 Kb were amplified by using twelve selected primers. The cluster analysis indicated that the eight varieties formed two major clusters.The first major cluster had only one variety ‘S-558’ with 43% similarity with other seven varieties. Second major cluster having seven varieties and divided into two minor clusters. One minor cluster had six varieties (‘Jugijan’, ‘Turia local’, ‘Nadia’, ‘Zo-17’, ‘Nahfrey’ and ‘Gurubathan) and other having only one variety ‘ Surabhi’. The second minor cluster further divided into two sub-minor clusters. ‘Nadia’ and ‘ZO-17’ had 78% similarity among themselves and 70% similarity with ‘Jugijan’ and ‘Turia local’. ‘Jugijan’ and ‘Turia local’ were having 81% similarity among themselves. However, ‘Nahfrey’ had 64% similarity with ‘Jugijan’, ‘Turia local’, ‘Nadia’ and ‘ZO-17’. The present study showed the distant variation within the varieties. This investigation will help to breeders for ginger improvement program.
Nicotine is synthesized in the root of tobacco (Nicotiana tabacum) and transported to aerial parts where this alkaloid functions as an insect repellant. Nicotine biosynthesis is regulated by jasmonate and specific regulatory Nic loci, but molecular components in the jasmonate- or Nic-dependent signaling pathways have not been well characterized. We here identified a tobacco gene encoding a novel protein JEI1 (Jasmonate Early Inducible 1) containing a BTB domain and a WD40-like repeat. The JEI1 expression was regulated by the NIC loci. In the leaf, application of jasmonate or wounding rapidly and transiently induced the JEI1 expression, with an induction pattern distinct from a wound-inducible protein kinase WIPK. This tobacco gene might be involved in wound-elicited signaling pathways.
Adventitious shoots were regenerated from leaf explants of 4 cultivars of auricula (Primula×pubescens Jacq.) on media supplemented with various cytokinins combined with NAA. One to four adventitious shoots per leaf explant were regenerated. The highest frequency of adventitious shoot formation was 46.6% for ‘Borders Mixed’ on the medium containing 0.2 mg l−1 TDZ and 2 mg l−1 NAA , 10% for ‘Border Stripes’ on the medium containing 0.2 mg l−1 TDZ and 2 mg l−1 NAA, 20% for ‘Alpines Mixed’ on the medium containing 0.02 mg l−1 TDZ and 2 mg l−1 NAA, 13.3% for ‘Field House Mixed’ on the medium containing 0.02 mg l−1 TDZ and 2 mg l−1 NAA. A higher concentration of NAA (2 mg l−1) combined with TDZ in the medium may stimulate adventitious shoot formation from leaf explants in auricula cultivars. Zeatin was less effective for adventitious shoot regeneration from leaf explants. Regenerated shoot easily rooted on plant growth regulator-free LS medium. After acclimatization in a growth chamber, some of the regenerated plants flowered.
In Vanda orchids, it is important to produce cultivars with economically important traits such as disease and pest resistances and novel flower colors, which are difficult to achieve by conventional cross breeding methods. To realize these breeding objectives, it is now expected to apply genetic transformation technology to introduce useful foreign genes into Vanda orchids. However, there has been almost no information on the genetic transformation of Vanda. Transgenic plants were successfully regenerated after co-cultivating protocorm-like bodies (PLBs) with Agrobacterium tumefaciens strain EHA101 (pIG121Hm) that harbored genes for β-glucuronidase (gus), hygromycin phosphotransferase (hpt) and neomycin phosphotransferase II (nptII). PLBs of ‘Tokyo Blue’ maintained in liquid New Dogashima medium (NDM) under dark condition, were subjected to transformation experiments. The PLBs inoculated with Agrobacterium produced secondary PLBs 4 weeks after transfer onto 3 g l−1 gellan gum-solidified NDM containing 30 g l−1 maltose, 10 mg l−1 meropenem and 10 mg l−1 hygromycin. Transformation efficiency was increased by prolonged period of infection (240 min) and wounding treatment of PLBs by sonication (5 min) during inoculation. Transformation of the hygromycin-resistant plantlets regenerated from different PLBs was confirmed by histochemical GUS assay, PCR analysis and Southern hybridization. With this transformation system, approximately 17 independent transgenic plants were obtained from 1 g PLBs.
To examine the capacity for plant regeneration through somatic embryogenesis, Japanese soybean cultivars were investigated by 3-step-somatic embryogenesis: 1) induction of somatic embryos (SEs) from immature cotyledons, 2) proliferation of SEs in liquid medium and 3) differentiation of SEs into cotyledon-stage embryos. At each step, properties of the cultures varied among cultivars. Notably, significant differences were observed on 1) efficiencies of SE induction, 2) compactness of individual SEs during proliferation, and 3) yields of differentiated embryos (numbers of cotyledon-stage embryos differentiated from 100 mg globular SEs). We found that the compactness of SEs in liquid medium was very important factor for the recovery of well differentiated embryos. In fact, Yuuzuru and Yumeyutaka which indicated high ratios of compact SEs at proliferation step showed high yields of well differentiated embryos. The abilities of these Japanese cultivars to yield cotyledon-stage embryos were superior or comparable to North American cultivar ‘Jack’, which is used as one of the most preferable genotypes for somatic embryogenesis, indicating their high potentials for plant regeneration.