Although mitochondria are known to move actively in plant cells, little is known about how they move. In higher plants, actin filaments have been reported to be involved in the movement of organelles, such as chloroplasts, peroxisomes, endoplasmic reticulum and Golgi apparatus. Mitochondria were visualized in living Arabidopsis thaliana plants using fluorescent proteins fused to a mitochondria targeting signal. To compare the movement of mitochondria to the movements of another organelle, we also examined peroxisomes because of their similarity in size. Velocities of individual mitochondria and peroxisomes, as measured by time-lapse laser scanning microscopy, varied, although the average velocities of the two organelles were similar. Latrunculin B, an actin-depolymerizing drug, stopped movement of mitochondria and peroxisomes, demonstrating that movement of these organelles depends on actin filaments. On the other hand, propyzamide, a microtubule-depolymerizing drug, did not affect the movement of mitochondria and peroxisomes. In living Arabidopsis plants in which mitochondria or peroxisomes are visualized by red fluorescent protein and cytoskeletal elements are simultaneously visualized by green fluorescent protein, both organelles moved using actin filaments as rails. In contrast, their movement was not related to arrays of microtubules. We also examined mitochondrial movement in tobacco cultured cells visualizing mitochondria and cytoskeletal element simultaneously. Mitochondrial movement was often seen along actin filaments but not along microtubules. These findings suggest that mitochondria move along actin filaments rapidly and over long distances in higher plants.
Endocytosis is an essential phenomenon in eukaryotic cells. In animal cells, dynamin and clathrin play central roles in vesicle formation in the process of endocytosis, but the roles of similar proteins in plants are less well understood. Here, we observed the localization pattern and behavior of GFP-labeled Arabidopsis dynamin-related proteins (DRP1A and DRP2B), and clathrin light chain (AtCLC) around the plasma membrane in tobacco suspension cells by using variable incidence angle fluorescence microscopy (VIAFM). GFP fusions of DRP1A, DRP2B and AtCLC were observed as dot-like puncta 200–500 nm in diameter. The puncta moved to and away from the cell surface or also assembled and disassembled. The localization pattern and behavior of the puncta were similar to those of animal dynamin and clathrin signals reported previously. These results raise the possibility that DRP1A, DRP2B and AtCLC are involved in membrane trafficking around the plasma membrane, including endocytosis.
In Arabidopsis, two myb-related proteins, LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), play key roles in the control of circadian rhythms. Photoperiodic flowering and elongation of hypocotyls are regulated by a circadian clock. Here, we report the isolation and characterization of a novel deletion allele (lhy-14) and a gain-of-function allele (lhy-2) of lhy. Based on long hypocotyl and late flowering phenotypes under long days, lhy-2 was isolated as an intragenic suppressor of lhy-12, one of the loss-of-function alleles of lhy. Although the lhy-12 mRNA has a 19-bp truncation, we found that this was not caused by a deletion in the LHY but by a point mutation. We propose a model explaining the loss-of-function of LHY in lhy-12 and the partial suppression of the lhy-12 phenotype by lhy-2, by the abnormal splicing of LHY mRNA.
In order to establish a system for characterizing gene function utilizing information obtained from genome sequences, we generated T-DNA insertion lines using a newly constructed binary vector. The vector carries a uidA [β-glucuronidase (GUS)] reporter gene which allows the promoter activity of the inserted genes to be monitored, a transposable element Dissociation (Ds) for targeted insertional mutagenesis, and the cis sequences required for Agrobacterium-mediated transformation. Approximately 8% of the 20,000 lines tested for GUS activity exhibited positive staining. Staining was detected in various organs including roots, leaves, stems, flowers, and siliques. These lines are therefore useful resources for analyzing tissue or organ specific gene expression. We have included the GUS expression patterns on our web site (http://www.kazusa.or.jp/ja/plant/GUS/). To establish a system for targeted insertional mutagenesis, integration sites of the T-DNAs in 140 lines with a single T-DNA insertion were mapped on the genome. The T-DNA contains the Ds element; therefore these 140 lines could be used as donor loci for Ds transposition. The integration sites were almost evenly distributed on all five chromosomes except for the nuclear organizer regions of chromosomes 2 and 4 and the centromeric regions. The Ds element in one line was transposed in combination with an Activator (Ac) element. In about half of the transposed lines, the Ds elements were reinserted within about 1 M bp from the donor locus. These results indicate that the donor loci for Ds transposition that were mapped in this study are a valuable resource for targeted mutagenesis throughout the Arabidopsis genome.
The Arabidopsis ESR1, which encodes a member of the ERF family, is thought to be a key gene for commitment to shoot differentiation in tissue culture. The Arabidopsis genome has more than a hundred genes encoding the ERF family and ESR1 belongs to the ERF VIII-b subgroup. We examined ESR1 and 5 genes structurally similar to ESR1 (ESR2, ESL1, ESL2, ESL3 and LEP) on their expression patterns during in vitro shoot regeneration. All these genes' transcript levels increased 1–2 days after shoot induction by incubation on shoot-inducing medium and then decayed by day 7. These genes' overexpression demonstrated that ESR1 and ESR2 clearly enhanced shoot regeneration when overexpressed, but other genes did not. These results suggest that all 6 examined genes may be involved in early events of shoot regeneration, although only ESR1 and ESR2 enhanced shoot regeneration by their overexpression.
In vitro protein phosphorylation in soluble fractions (85,000×g supernatant) prepared from carrot hairy roots, which were induced by inoculation with Agrobacterium rhizogenes harboring the Ri plasmid, was analyzed by SDS-PAGE and autoradiography. The autoradiograms indicated that at least four proteins, with molecular masses of 65, 57.5, 52, and 38 kDa, were phosphorylated. The presence of Ca2+ enhanced the phosphorylation of the 65, 57.5, and 52 kDa proteins, but the presence of both Ca2+ and calmodulin did not enhance phosphorylation as much as Ca2+ alone. W-7 strongly inhibited the phosphorylation of the 65, 57.5, and 52 kDa proteins while W-5 mildly inhibited the phosphorylation of these proteins. The extent of phosphorylation of these proteins and the growth rate of the hairy roots became higher with increasing incubation temperature. W-7 had a considerable inhibitory effect on the growth of hairy roots, W-5 had a weak inhibitory effect on the growth of the roots, and a high concentration of TMB-8 inhibited growth. These results suggest that the 65, 57.5, and 52 kDa proteins are phosphorylated by CDPK and that the phosphorylation is connected with the increase in growth of carrot hairy roots.
Some polyacylated anthocyanins, anthocyanins containing two or more aromatic acyl groups, are blue in color within a wide range of pH values, including acidic and neutral conditions even in the absence of any co-pigments or metal ions. We engineered a mutant of a malonyl-CoA:anthocyanin 5-O-glucoside-6″′-O-malonyltransferase of Salvia splendens (Ss5MaT1), Ss5AT306, which has an acquired specificity for hydroxycinnamoyl-CoA and is able to produce novel polyacylated anthocyanins. Ss5AT306 showed 6″′-O-hydroxycinnamoyltransferase activity toward some anthocyanins in addition to the 6″′-O-malonyltransferase activity retained (relative activities for acyl transfer to shisonin: malonyl-CoA, 100%; p-coumaroyl-CoA, 132%; caffeoyl-CoA, 103%). This alteration of acyl-donor specificity was achieved by the substitutions of only three contiguous amino acid residues, Val39-Arg40-Arg41, to the corresponding residues of anthocyanin aromatic acyltransferases, Met-Leu-Gln, suggesting that these amino acid residues are key residues governing the malonyl-CoA specificity of Ss5MaT1. Through the use of Ss5AT306, a novel polyacylated anthocyanin, p-coumaroylshisonin, with one aromatic acyl group in each of the 3-O-glucosyl and 5-O-glucosyl moieties of its structure, was produced and characterized in vitro. p-Coumaroylshisonin was bluer in color and displayed stronger color intensity than did shisonin, implying that accumulation of polyacylated anthocyanins such as p-coumaroylshisonin cause the modulation of flower colors.
Methyl chloride and methyl bromide, which contribute to the destruction of the stratospheric ozone layer, are mainly emitted from natural sources. It was recently reported that tropical and subtropical plants were the largest sources of methyl chloride. Furthermore, the involvement of the gene HARMLESS TO OZONE LAYER (HOL) in methyl halide emissions from Arabidopsis thaliana was demonstrated. However, neither the physiological significance of the methyl chloride emission nor the biochemical properties of HOL, denoted as AtHOL1 in our study, have been reported yet. We identified two additional isoforms-AtHOL2 and AtHOL3-from Arabidopsis and characterized them together with AtHOL1. AtHOL1 was ubiquitously expressed during development, and its expression level was the highest among the three. The phylogenetic tree suggested that AtHOL1 homologous proteins were distributed throughout the plant kingdom. Biochemical analyses showed that the three recombinant AtHOL proteins were functional and had distinct levels of the S-adenosyl-L-methionine-dependent methyltransferase activities. Although a study of AtHOL1-disrupted mutants had shown that AtHOL1 primarily controlled the production of methyl halide, our study suggested that the activation of AtHOL2 and AtHOL3 genes also contribute to the methyl halide emissions from Arabidopsis.
The possibility of developing transgenic indica rices through Agrobacterium-mediated transformation in the absence of acetosyringone at bacterial preinduction or co-cultivation or both stages was assessed. Four-week-old, scutellum derived calluses of indica rice (Oryza sativa L. cv. ‘Pusa Basmati1’) were co-cultivated with A. tumefaciens strain LBA4404 (pSB1), harboring the binary vector pCAMBIA 1301 with the β-glucuronidase (GUS) and hygromycin phosphotransferase (HPT) genes in the T-DNA region. Addition of acetosyringone (AS) to both preinduction medium (PIM) and co-cultivation medium (CCM) induced higher levels of transient GUS expression than that obtained with the addition of AS to either of the stages. Addition of only sucrose to both preinduction and co-cultivation media yielded transient expression levels similar to those obtained by the addition of AS. The resultant fertile plants were stable transformants as revealed by GUS histochemical assay and PCR analysis for the GUS and HPT genes. Thus, phenolics like AS may not be essential for induction of vir genes, and development of transgenic indica rice is feasible under AS-free conditions.
Gibberellins (GAs) are involved in the expression of cysteine proteinase genes in germinated cotyledons of common bean seeds. Because DELLA proteins are known to be transcriptional repressors mediating GA signaling, we isolated two cDNA clones encoding DELLA proteins (PvGAI1 and PvGAI2) from common bean seedlings to examine the mechanism of GA signaling involved in the expression of the proteinase genes. RT-PCR and RNA blot analyses indicated that the level of mRNA in germinated cotyledons was higher for PvGAI2 than for PvGAI1. We also found that transient expression of PvGAI2, but not that of PvGAI1, repressed the promoter activities of GA-inducible cysteine proteinase genes, EP-C1 and CP2, in germinated cotyledons. These findings suggest that PvGAI2 is mainly responsible for regulating the expression of proteinase genes in germinated cotyledons. Application of a GA-biosynthesis inhibitor, prohexadione calcium to common bean seeds had little effect on the RNA level of PvGAI2, although the inhibitor repressed genes for EP-C1 and CP2. Because it is known that GA induces degradation of DELLA proteins, our findings suggest that the level of GA, but not the mRNA expression of PvGAI2, regulates the protein level of PvGAI2 suppressing the proteinases genes in germinated cotyledons.
The species/varieties of Polyscias and Schefflera are important ornamental foliage pot plants. The germplasm identification and characterization is an important link between the conservation and utilization of plant genetic resources. Investigation were undertaken for identification and determination of genetic variation within 14 species/varieties of Polyscias and one species of Schefflera elegantissima under family Araliaceae through RAPD (random amplified polymorphic DNA) and ISSR (Inter Simple Sequence Repeats) markers. Genetic analysis was made by using 15 selected decamer primers and 9 selected ISSR markers. A total of 164 and 69 distinct DNA fragments ranging from 300 to 2500 bp were amplified by using selected random RAPD and ISSR primers respectively. The genetic similarity was evaluated on the basis of presence or absence of bands. The cluster analysis was made by using similarity coefficient. The cluster analysis indicated that the 14 varieties/species of Polyscias formed one major cluster and Schefflera elegantissima forming another major cluster. There were distant variation among the genus or species and close variation was obtained among the varieties of Polyscias. The correlation matrix indicates that there was significant correlation between ISSR and RAPD markers. Thus, these markers have the potential for identification of species/varieties and variation within the varieties. This is also helpful in breeding programs as well as a major input into conservation biology of foliage crop.
In this study we have investigated micropropagation and in vitro flowering for a medicinally important plant Pentanema indicum. Maximum callus proliferation was obtained on MS medium supplemented with 2.0 mg l−1 BAP and 1.0 mg l−1 IBA. The best shoot regeneration (19±1.0) was achieved in five weeks when callus was cultured on MS medium amended with 4.0 mg l−1 BAP and 1.0 mg l−1 IAA. Direct multiple shoot initiation was also obtained from shoot tip and nodal explants in the presence of BAP and IAA. Addition of adenine sulfate (1.0 mg l−1) to the regeneration medium increased the shoot multiplication. Regenerated shoots rooted best on MS medium containing 2.0 mg l−1 IBA. Multiple shoots regenerated from callus, shoot tip and nodal explants flowered (90%) in vitro on a MS medium fortified with 2.0 mg l−1 IBA. Plantlets were successfully acclimatized in a soil condition and the survival rate was 96%.
A soybean variety, Kariyutaka was found to form much more precocious flower buds than those of the other six genotypes used in this study when in vitro cultured at 26°C with 16/8 h light/dark regime (fluorescent tubes at an intensity of about 100 μmols s−1 m−2). In addition, it was a suitable genotype for soybean transformation because it was highly sensitive to Agrobacterium and it exhibited excellent shoot regeneration. Its transformation frequency (about 1%) was comparable to that of a variety Thorne, which is known as one of the suitable cultivars for Agrobacterium-mediated transformation. Because transgenic shoots of Kariyutaka formed in vitro precocious flower buds, T1 seeds were obtained in only six months after co-cultivation with Agrobacterium. A few copy numbers of transgenes were inheritable to T1 generations. T1 plants also produced seed within 2–3 months when grown at the same regime as in vitro culture. Hence Agrobacterium-mediated transformation using Kariyutaka can be recommended as a model method for the rapid development of transgenic soybean lines.