Agrobacterium-mediated transformation of Indian mulberry, Morus indica cv. K2, employing the highly regenerative hypocotyl, cotyledon, leaf and leaf callus explants is reported. Preculture of explants on regeneration medium for 5 days and co-cultivation for 3 days was found optimal. With the GUS-histochemical assay up to 40% hypocotyl, 50% cotyledon, 50% leaf and 54% leaf callus explants tested positive. Agrobacterium strain LBA4404 was more infective than GV2260 and A281 and amongst the plasmids tested, pBI121 successfully transformed 100% of the explants followed by p35SGUSINT (90-100%). Approximately, 50% of the explants survived selection. Up to 35.7% hypocotyl, 38.5% cotyledon, 23% leaf and 40% leaf callus derived shoots tested positive for GUS gene activity by the spectrofluorometric analysis. Transgene integration in the regenerated transformants was confirmed by polymerase chain reaction (PCR) and Southern hybridization making this the first report of stable transformation of mulberry.
Antagonistic interaction between endogenous sugar status and abscisic acid (ABA) signaling was examined in Arabidopsis. Endogenous ABA content in suspension-cultured cells and seedlings grown in the dark increased following reduction in endogenous sugar content. Expression of Arabidopsis 9-cis epoxycarotenoid dioxygenase (AtNCED ) genes, which encode key enzymes for ABA biosynthesis and ABSCISIC ACID INSENSITIVE 3 (ABI 3) which controls ABA sensitivity was enhanced by sugar starvation. GUS activity analysis in transgenic Arabidopsis carrying GUS controlled by the ABI3 promoter showed that ABI3 was expressed, when sugar was depleted and thereby leaf development was terminated, in the shoot apical meristem. We conclude that sugar depletion promotes ABA signaling to terminate leaf development in Arabidopsis seedlings. Antagonistic effects of sugar on ABA signaling in the process of vegetative quiescence processs are discussed.
The effects of two-intron insertions on reporter-gene expression were examined in transient assays in maize and tobacco. The first introns of a rice gene for phospholipase D (PLD), a maize gene for ubiquitin and a castor bean gene for catalase were tested with a gene for β-glucuronidase (GUS) or a gene for luciferase driven by a cauliflower mosaic virus 35S promoter (35S-GUS or 35S-Luc), the promoter of the maize ubiquitin (Ubi-GUS) or the promoter of a maize pyruvate, orthophosphate dikinase (PPDK-GUS). Although the enhancing effect varied, several intron combinations synergistically enhanced the reporters. When the first and second introns of the PLD gene were inserted in tandem, the reporter was synergistically enhanced. These results imply that expression enhancement by two introns may be observed in wide combinations of promoters, introns, and structural genes and that such enhancement plays certain roles in regulation of gene expression in higher plants.
A simple and efficient method for somatic embryogenesis of chrysanthemum [Dendranthema × grandiflorum (Ramat.) Kitamura] was established. The best result of somatic embryogenesis was obtained with MS medium containing 2.0 mg l-1 2, 4-dichlorophenoxyacetic acid and 1.0 mg l-1 kinetin. Embryos with roots 1 to 2 mm long gave the highest frequency for conversion of embryos to plantlets. Root meristems appeared to be formed in somatic embryos by 20 days after culture initiation, while the shoot apices formed after replanting in phytohormone-free medium, resulting in a bipolar structure. The embryo-specific ECP63 gene was expressed in the somatic embryos, as in zygotic embryos, confirming that the plant regeneration occurred via embryogenesis. The growth habits and leaf and flower morphology of the regenerated plantlets were normal, but the day of flowering differed from that of cutting controls. The present somatic embryogenesis method was applicable to 8 out of 13 cultivars studied.
A direct phosphorylation screening of a rice cDNA library resulted in isolation of 35 BIP clones encoding brassinosteroid receptor kinase (BRI1)-interacting proteins. Among the candidate substrates for BRI1, two clones were found to encode similar proton pump interactor proteins homologous to Arabidopsis PPI1, which was reported to interact with a regulatory region of plasma membrane H+-ATPase. The rice proton pump interactors BIP103 and BIP131 contained 627 and 621 amino acids, respectively, with carboxyl-terminal hydrophobic region characteristic of tail-anchored proteins. Northern blotting analysis indicated that mRNAs for both interactors increased significantly after brassinolide treatment of lamina joint cells, which are especially sensitive to exogenous brassinosteroids.
Endopeptidase activities during in vitro tracheary element (TE) differentiation of Zinnia cells were investigated using peptidyl 4-methylcoumaryl-7-amide (MCA) compounds as substrate. Endopeptidase activities against Boc-Val-Leu-Lys-MCA, with an acidic pH optimum and against Boc-Phe-Ser-Arg-MCA, with an alkaline pH optimum were found to be induced preferentially in cells cultured in TE differentiation-inductive medium, although the former activity appeared earlier than the latter one during the culture. Each activity was eluted as a single peak in DEAE-Sepharose column chromatography. The nature of Boc-Val-Leu-Lys-MCA hydrolyzing activity was similar to that of a papain-like cysteine protease as reported previously by our group. On the other hand, the partially purified Boc-Phe-Ser-Arg-MCA hydrolyzing activity was completely inhibited by leupeptin and weakly by E-64, but not by pepstatin A, 1, 10-phenanthroline, or PMSF, suggesting that this activity results from a cysteine protease(s). These results reveal the Boc-Phe-Ser-Arg-MCA hydrolyzing activity is due to a novel cysteine protease with an alkaline pH optimum which may function during TE differentiation.
This study was carried out to trace the biological effects of exposing Agrobacterium tumefaciens and sugarcane cell clumps of suspension culture to ultrasonic waves. A diluted suspension of Agrobacterium tumefaciens, strains EHA101 and LBA4404, was treated with 44 kHz sonication which remarkably reduced the frequency of colony formation in the inoculated medium of both strains. The cell clumps of the suspension culture were co-cultured with Agrobacterium, and then sonicated. Following 3 days repeating co-culture in solid medium, the cell clumps were transferred to the MS-1e medium. Without sonication treatment, 31% of cell clumps revealed bacterial overgrowth, whereas no overgrowth was found in the sonication treatment after 14 days of culture. However, no significant differences in the rates of growth and shoot regeneration between the sonication-treated cell clumps and the untreated cell clumps.
Vectors with two separate T-DNAs for co-transformation mediated by Agrobacterium tumefaciens were tested in maize inbred A188. Although the frequency of transformation during the initial trials was very low, it was remarkably improved by the vector with a phosphinothricin resistance gene (bar) using a modified transformation protocol, which is characterized by the presence of silver nitrate and carbenicillin in the selection medium. The vector with hygromycin resistance gene (hpt) showed low frequency even with the improved protocol, but the use of maize ubiquitin gene promoter elevated the frequency. Both co-transformation and segregation of the progeny free from the selection markers frequencies were reasonably high and similar to those previously observed in rice and tobacco. The two-T-DNA vectors will be a useful tool in molecular biology and biotechnology studies of maize.
DNA damage recognition during nucleotide excision repair (NER) involves the homologous heterodimers Rad4:Rad23 in budding yeast and XPC:hHR23B in human. We report here the characteristics of four Arabidopsis homologues of RAD23 gene, named AtRAD23-1 to-4. AtRAD23-1, -3 and -4 expressed two alternatively spliced transcripts, long ones (AtRAD23-1α, -3α and -4α) and short ones (AtRAD23-1β, -3β and -4β). The predicted amino acid sequences of these genes possessed four conserved domains of Rad23 family; the ubiquitin-like domain, ubiquitin-associated domain I, XPC-binding domain and ubiquitin-associated domainII. AtRad23-3 βand-4 βlacked the C-terminus ubiquitin-like domain and the C-terminus XPC-binding domain, respectively, suggesting that these alternatively spliced variants may modulate functional AtRad23 proteins. Phylogenetic analysis showed that plant RAD23 genes could be divided into two classes and that Arabidopsis RAD23 genes were recently duplicated. AtRAD23-1-4 transcripts were detected in various tissues, with the highest expression level in flower buds.
In Spinacia oleacea, three isoforms of cysteine synthase (CSase, O-acetyl-serine (thiol) lyase) were identified in the cytosolic, plastidial, and mitochondorial compartments. Here, we report molecular cloning of a cDNA that encodes a CSase-like protein, designated CSaseLP. The predicted amino acid sequence of CSaseLP showed high identity (about 70%) with that of a spinach cytosolic CSase (encoded by CSaseA gene). The CSase activity of recombinant CSaseLP was barely detectable. The antibody raised against the recombinant CSaseLP protein recognized weakly a recombinant CSaseA protein. Northern blot analysis showed that CSaseLP gene was expressed mainly in root tissues, while the expression of CSaseA gene was constitutive in leaf and root tissues. These results suggest that the CSaseLP protein, though belongs to the CSase family, play a role distinct from the cytosolic CSase.
Homoglutathione (hGSH) is present in some leguminous plants including soybean and bean. However, hGSH synthesis in non-leguminous plants has not been reported so far. We constructed transgenic tobacco plants expressing soybean hGSH synthetase (hGSHS). Although the transgenic tobacco plants exhibited higher hGSHS activity than glutathione (GSH) synthetase activity, hGSH content was much lower than GSH content. Precursor feeding experiments revealed that hGSH synthesis was limited by the availability of both γ-L-glutamyl-L-cysteine and β-alanine. The result also suggests that the major site of GSH synthesis in tobacco plants is not cytosol.