Reorganized actin filaments anchor chloroplasts along the anticlinal walls of <Emphasis Type="Italic">Vallisneria</Emphasis> epidermal cells under high-intensity blue light

In epidermal cells of the aquatic angiosperm Vallisneria gigantea Graebner, high-intensity blue light (BL) induces the avoidance response of chloroplasts. We examined simultaneous BL-induced changes in the configuration of actin filaments in the cytoplasmic layers that face the outer periclinal wall (P side) and the anticlinal wall (A side). The results clearly showed that dynamic reorganization of the actin cytoskeleton occurs on both sides. Upon BL irradiation, thick, long bundles of actin filaments appeared, concomitant with the directed migration of chloroplasts from the P side to the A side. After 15–20 min of BL irradiation, fine actin bundles on only the A side appeared to associate with chloroplasts that had migrated from the P side. To examine the role of the fine actin bundles, we evaluated the anchorage of chloroplasts by centrifuging living cells. Upon BL irradiation, the resistance of chloroplasts on both the P and A sides to the centrifugal force decreased remarkably. After 20 min of BL irradiation, the resistance of chloroplasts on the A side increased again, but chloroplasts on the P side could still be displaced. The BL-induced recovery of resistance of chloroplasts on the A side was sensitive to photosynthesis inhibitors but insensitive to an inhibitor of flavoproteins. The photosynthesis inhibitors also prevented the fine actin bundles from appearing on the A side under BL irradiation. These results strongly suggest that the BL-induced avoidance response of chloroplasts includes photosynthesis-dependent and actin-dependent anchorage of chloroplasts on the A side of epidermal cells.     

Regulation of the orientation movement of chloroplasts in epidermal cells of Vallisneria: cooperation of phytochrome with photosynthetic pigment under low-fluence-rate light

In epidermal cells of the leaves of the aquatic angiosperm Vallisneria gigantea Graebner, the chloroplasts accumulate in the outer periclinal layer of cytoplasm (P side) under light at low fluence rates. The nature of such intracellular orientation of chloroplasts was investigated in a semiquantitative manner. Time-lapse video microscopy revealed that, while irradiation with red light (650 nm, 0.41 W · m^–2) rapidly accelerated the migration of chloroplasts, not only from the anticlinal layers of cytoplasm (A sides) to the P side but also from the P side to the A sides, the increased rate of migration in both directions returned to the control rate upon subsequent irradiation with far-red light (746nm, 0.14W · m^–2). These effects of red and far-red light could be observed repeatedly, both in the presence and in the absence of inhibitors of photosynthesis, suggesting the involvement of phytochrome as the photoreceptor. After saturating irradiation with red light, the increased rate of migration of chloroplasts from the P side to the A sides declined more rapidly than the increased rate of migration in the opposite direction. This imbalance in the migration of chloroplasts between the two opposing directions resulted in the accumulation of chloroplasts on the P side. The more rapid decline in the rate of migration of chloroplasts from the P side to the A sides than in the opposite direction was not observed in the presence of an inhibitor of photosynthesis. It appears, therefore, that phytochrome and photosynthetic pigment cooperatively regulate the accumulation of chloroplasts on the P side through modulation of the nature of the movement of the chloroplasts.Abbreviations A side cytoplasmic layer that faces the anticlinal wall - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Pfr farred-light-absorbing form of phytochrome - Pr red-light-absorbing form of phytochrome - P side cytoplasmic layer that faces the outer periclinal wall This work was supported in part by Grants-in-Aid from the Japanese Ministry of Education, Science and Culture to S.T. and R.N. The authors are indebted to the Osaka branch of Kashimura Inc. for their kind cooperation in preparing the GREEN software.     

Altered expression of the<Emphasis Type="Italic"> Arabidopsis</Emphasis> ortholog of<Emphasis Type="Italic"> DCL</Emphasis> affects normal plant development

The DCL (defective chloroplasts and leaves) gene of tomato (Lycopersicon esculentum Mill.) is required for chloroplast development, palisade cell morphogenesis, and embryogenesis. Previous work suggested that DCL protein is involved in 4.5S rRNA processing. The Arabidopsis thaliana (L.) Heynh. genome contains five sequences encoding for DCL-related proteins. In this paper, we investigate the function of AtDCL protein, which shows the highest amino acid sequence similarity with tomato DCL. AtDCL mRNA was expressed in all tissues examined and a fusion between AtDCL and green fluorescent protein (GFP) was sufficient to target GFP to plastids in vivo, consistent with the localization of AtDCL to chloroplasts. In an effort to clarify the function of AtDCL, transgenic plants with altered expression of this gene were constructed. Deregulation of AtDCL gene expression caused multiple phenotypes such as chlorosis, sterile flowers and abnormal cotyledon development, suggesting that this gene is required in different organs. The processing of the 4.5S rRNA was significantly altered in these transgenic plants, indicating that AtDCL is involved in plastid rRNA maturation. These results suggest that AtDCL is the Arabidopsis ortholog of tomato DCL, and indicate that plastid function is required for normal plant development.Abbreviations DCL Defective chloroplasts and leaves - GFP Green fluorescent protein     

Biochemical and immunohistochemical characterization of<Emphasis Type="Italic"> Mimosa</Emphasis> annexin

To characterize the biochemical properties of plant annexin, we isolated annexin from Mimosa pudica L. and analyzed the biochemical properties conserved between Mimosa annexin and animal annexins, e.g. the ability to bind phospholipid and F-actin in the presence of calcium. We show that Mimosa annexin is distributed in a wide variety of tissues. Immunoblot analysis also revealed that the amount of annexin is developmentally regulated. To identify novel functions of Mimosa annexin, we examined the pattern of distribution and the regulation of its expression in the pulvinus. The amount of annexin in the pulvinus increased at night and was sensitive to abscisic acid; however, there was no detectable induction of annexin by cold or mechanical stimulus. Annexin distribution in the cell periphery during the daytime was changed to a cytoplasmic distribution at night, indicating that Mimosa annexin may contribute to the nyctinastic movement in the pulvinus.Abbreviations ABA Abscisic acid - PC Phosphatidylcholine - PS Phosphatidylserine     

Antibacterial activity of<Emphasis Type="Italic">Ulva lactuca</Emphasis> against methicillin-resistant<Emphasis Type="Italic">Staphylococcus aureus</Emphasis> (MRSA)

Thein vitro antimicrobial activity of the marine green algaeUlva lactuca was examined against gram-positive bacteria, gram-negative bacteria, and a fungus. The ethyl-ether extract of algae exhibited a broad-spectrum of antibacterial activity. but not antifungal activity againstCandida albicans. In particular, theU. lactuca extract showed strong activity aganst the bacterium methicillin-resistantStaphylococcus aureus (MRSA). This result confirms the potential use of seaweed extracts as a source of antibacterial compounds or as a health-promoting food for aquaculture.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-<Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of<Emphasis Type="Italic"> Helminthosporium turcicum</Emphasis>, the maize leaf-blight fungus

Agrobacterium tumefaciens has the ability to transfer its T-DNA to plants, yeast, filamentous fungi, and human cells and integrate it into their genome. Conidia of the maize pathogen Helminthosporium turcicum were transformed to hygromycin B resistance by a Agrobacterium-tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase (hph) and the enhanced green fluorescent protein (EGFP) genes controlled by the gpd promoter from Agaricus bisporus and the CaMV 35S terminator. Agrobacterium-tumefaciens-mediated transformation yielded stable transformants capable of growing on increased concentrations of hygromycin B. The presence of hph in the transformants was confirmed by PCR, and integration of the T-DNA at random sites in the genome was demonstrated by Southern blot analysis. Agrobacterium-tumefaciens-mediated transformation of Helminthosporium turcicum provides an opportunity for advancing studies of the molecular genetics of the fungus and of the molecular basis of its pathogenicity on maize.     

Light-susceptibility of camptothecin production from<Emphasis Type="Italic">in vitro</Emphasis> cultures of<Emphasis Type="Italic">Camptotheca acuminata</Emphasis> Decne

Production of camptothecin (CPT) from callus cultures ofCamptotheca acuminata Decne was affected by light and culture conditions. Among the culture media tested, modified B5 medium containing 3% (w/v) sucrose, 2 mg/L 2,4-D, 2 times of MS medium vitamins, 500 mg/L casein hydrolysate, 250 mg/L myo-inositol, 0.05% (w/v) activated charcoal, and 0.15% (w/v) gelite was used for callus induction. The highest cell growth and CPT production were obtained in dark and green light condition, respectively. Photoperiod has no effect on cell growth and CPT production. Both cell growth and CPT production were also influenced by combination ratio of red and blue light. Cell growth and CPT production were the highest in the ratio of red and blue light 90∶10.     

Behaviour of the hyphae of<Emphasis Type="Italic"> Laccaria laccata</Emphasis> in the presence of<Emphasis Type="Italic"> Trichoderma harzianum</Emphasis> in vitro

The growth rate and the behaviour of Laccaria laccata and Trichoderma harzianum hyphae in co-culture and in the rhizosphere of 3-month-old Pinus sylvestris seedlings grown in vitro were investigated. In the interaction zone, hyphae of L. laccata became more pigmented and formed short branches growing towards the hyphae of the saprobic fungus, coiled around them and penetrated sporadically. Vacuolated hyphae of T. harzianum showed protoplasm granulation and breaks in walls followed by release of protoplasts. In the rhizosphere, the mantle hyphae of L. laccata showed a tendency to surround conidia of T. harzianum. No obvious penetration of the conidial walls by the hyphae of the mycorrhizal fungus was observed by scanning electron microscopy. Instead, in rare cases, the hyphae of L. laccata showed marked wrinkles, and a partial degradation of a mucilaginous material covering the mantle appeared to occur.     

The<Emphasis Type="Italic"> GAOLAOZHUANGREN2</Emphasis> gene is required for normal glucose response and development of<Emphasis Type="Italic"> Arabidopsis</Emphasis>

Recent studies of glucose (Glc) sensing and signaling have revealed that Glc acts as a critical signaling molecule in higher plants. Several Glc sensing-defective Arabidopsis mutants have been characterized in detail, and the corresponding genes encoding Glc-signaling proteins have been isolated. However, the full complexity of Glc signaling in higher plants is not yet fully understood. Here, we report the identification and characterization of a new Glc-insensitive mutant, gaolaozhuangren2 (glz2), which was isolated from transposon mutagenesis experiments in Arabidopsis. In addition to its insensitivity to Glc, the glz2 plant exhibits several developmental defects such as short stature with reduced apical dominance, short roots, small and dark-green leaves, late flowering and female sterility. Treatment with 4% Glc blocked expression of the OE33 gene in wild-type plants, whereas expression of this gene was unchanged in the glz2 mutant plants. Taken together, our results suggest that the GLZ2 gene is required for normal glucose response and development of Arabidopsis.Mingjie Chen and Xiaoxiang Xia contributed equally to this work.     

Analyses of<Emphasis Type="Italic"> cis</Emphasis> -acting elements that affect the transposition of<Emphasis Type="Italic"> Mos1 mariner</Emphasis> transposons in vivo

The left (5) inverted terminal repeat (ITR) of the Mos1 mariner transposable element was altered by site-directed mutagenesis so that it exactly matched the nucleotide sequence of the right (3) ITR. The effects on the transposition frequency resulting from the use of two 3 ITRs, as well as those caused by the deletion of internal portions of the Mos1 element, were evaluated using plasmid-based transposition assays in Escherichia coli and Aedes aegypti. Donor constructs that utilized two 3 ITRs transposed with greater frequency in E. coli than did donor constructs with the wild-type ITR configuration. The lack of all but 10 bp of the internal sequence of Mos1 did not significantly affect the transposition frequency of a wild-type ITR donor. However, the lack of these internal sequences in a donor construct that utilized two 3 ITRs resulted in a further increase in transposition frequency. Conversely, the use of a donor construct with two 3 ITRs did not result in a significant increase in transposition in Ae. aegypti. Furthermore, deletion of a large portion of the internal Mos1 sequence resulted in the loss of transposition activity in the mosquito. The results of this study indicate the possible presence of a negative regulator of transposition located within the internal sequence, and suggest that the putative negative regulatory element may act to inhibit binding of the transposase to the left ITR. The results also indicate that host factors which are absent in E. coli, influence Mos1 transposition in Ae. aegypti.Communicated by G. P. Georgiev     

The<Emphasis Type="Italic"> Arabidopsis RHD3</Emphasis> gene is required for cell wall biosynthesis and actin organization

The Arabidopsis thaliana (L.) Heynh. ROOT HAIR DEFECTIVE3 (RHD3) gene has previously been shown to be essential for normal cell expansion [H. Wang et al. (1997) Genes Dev 11:799-811]. In this report, we demonstrated that mutation of the RHD3 gene in the Arabidopsis fragile fiber 4 (fra4) mutant caused a dramatic reduction in the wall thickness of fibers, vessels, and pith cells in the inflorescence stems and, concomitantly, a decrease in the mechanical strength of stems. The reduced wall thickness in the fra4 mutant was accompanied by an alteration in cell wall composition. Consistent with the defective fiber and vessel wall phenotypes, the RHD3 gene exhibited a strong expression in developing fiber and xylem cells. We showed that the Arabidopsis genome contains two additional RHD3-like genes, one of which was expressed specifically in flowers. In addition, we found that mutation of the RHD3 gene caused an alteration in the organization of the actin cytoskeleton but no effects on cortical microtubules. Our findings suggest an essential role of RHD3 in cell wall biosynthesis and actin organization, both of which are known to be important for cell expansion.     

Structural and transcriptional analysis of<Emphasis Type="Italic"> S</Emphasis>-locus F-box genes in<Emphasis Type="Italic"> Antirrhinum</Emphasis>

A class of ribonucleases termed S-RNases, which control the pistil expression of self-incompatibility, represents the only known functional products encoded by the S locus in species from the Solanaceae, Scrophulariaceae and Rosaceae. Previously, we identified a pollen-specific F-box gene, AhSLF (S locus F-box)-S₂, very similar to S₂-RNase in Antirrhinum, a member of the Scrophulariaceae. In addition, AhSLF-S₂ also detected the presence of its homologous DNA fragments. To identify these fragments, we constructed two genomic DNA libraries from Antirrhinum self-incompatible lines carrying alleles S₁S₅ and S₂S₄, respectively, using a transformation-competent artificial chromosome (TAC) vector. With AhSLF-S₂-specific primers, TAC clones containing both AhSLF-S₂ and its homologs were subsequently identified (S₂TAC, S₅TACa, S₄TAC, and S₁TACa). DNA blot hybridization, sequencing and segregation analyses revealed that they are organized as single allelic copies (AhSLF-S₂, -S₁, -S₄ and -S₅) tightly linked to the S-RNases. Furthermore, clusters of F-box genes similar to AhSLF-S₂ were identified. In total, three F-box genes (AhSLF-S₂, -S₂A and -S₂C) in S₂TAC (51 kb), three (AhSLF-S₄, -S₄A and -S₄D) in S₄TAC (75 kb), two (AhSLF-S₅ and -S₅A) in S₅TACa (55 kb), and two (AhSLF-S₁ and -S₁E) in S₁TACa (71 kb), respectively, were identified. Paralogous copies of these genes show 38–54% identity, with allelic copies sharing 90% amino acid identity. Among these genes, three (AhSLF-S₂C, -S₄D and -S₁E) were specifically expressed in pollen, similar to AhSLF-S₂, implying that they likely play important roles in pollen, whereas three AhSLF-SA alleles showed no detectable expression. In addition, several types of retroelements and transposons were identified in the sequenced regions, revealing some detailed information on the structural diversity of the S locus region. Taken together, these results indicate that both single allelic and tandemly duplicated genes are associated with the S locus in Antirrhinum. The implications of these findings in evolution and possible roles of allelic AhSLF-S genes in the self-incompatible reaction are discussed in species like Antirrhinum.Sequence data from this article have been deposited with the EMBL/GenBank databases under accession numbers AJ300474, AJ515534, AJ515536 and AJ515535     

An improved protocol for<Emphasis Type="Italic"> Agrobacterium</Emphasis>-mediated transformation of<Emphasis Type="Italic"> Antirrhinum majus</Emphasis> L.

Efficient Agrobacterium -mediated transformation of Antirrhinum majus L. was achieved via indirect shoot organogenesis from hypocotyl explants of seedlings. Stable transformants were obtained by inoculating explants with A. tumefaciens strain GV2260 harboring the binary vector pBIGFP121, which contains the neomycin phosphotransferase gene (NPT II) as a selectable marker and the gene for the Green Fluorescent Protein (GFP) as a visual marker. Putative transformants were identified by selection for kanamycin resistance and by examining the shoots using fluorescence microscopy. PCR and Southern analyses confirmed integration of the GFP gene into the genomes of the transformants. The transformants had a morphologically normal phenotype. The transgene was shown to be inherited in a Mendelian manner. This improved method requires only a small number of seeds for explant preparation, and three changes of medium; the overall transformation efficiency achieved, based on the recovery of transformed plants after 4–5 months of culture, reached 8–9%. This success rate makes the protocol very useful for producing transgenic A. majus plants.Communicated by G. Jürgens     

Expression of<Emphasis Type="Italic"> ENOD40</Emphasis> during tomato plant development

In legumes, ENOD40 expression is increased upon interaction of plants with rhizobia. Little is known of the expression pattern of ENOD40 during other stages of the plant life cycle. Studies of ENOD40 expression in non-legume development may give an indication of the function of the gene. To investigate the ENOD40 expression pattern during plant development, a fusion between the -glucuronidase (GUS) reporter gene and 150 bp of the 5 untranslated region plus 3,000 bp of 5 untranscribed tomato ENOD40 sequence was constructed and introduced into Lycopersicon esculentum Miller. Based on the observed GUS expression patterns in transgenic tomato we speculate that ENOD40 in tomato has a role in counteracting ethylene-provoked responses.Abbreviations GUS -glucuronidase - FISH fluorescence in situ hybridisation - RACE rapid amplification of cDNA ends - RFLC restriction fragment length polymorphism     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Thidiazuron Promotes <Emphasis Type="Italic">In Vitro</Emphasis> Plant Regeneration of <Emphasis Type="Italic">Arachis correntina</Emphasis> (<Emphasis Type="Italic">Leguminosae</Emphasis>) via Organogenesis

Arachis correntina (Burkart) Krapov. & W.C. Gregory is a herbaceous perennial leguminous plant growing in the Northeast of the Province Corrientes, Argentina. It is important as forage. The development of new A. correntina cultivars with improved traits could be facilitated through the application of biotechnological strategies. The purpose of this study was to investigate the plant regeneration potential of mature leaves of A. correntina in tissue culture. Buds were induced from both petiole and laminae on 0.7% agar-solidified medium containing 3% sucrose, salts and vitamins from Murashige and Skoog (MS) supplemented with 0.5–25 M thidiazuron (TDZ). Shoot induction was achieved by transference of calli with buds to MS supplemented with 5 M TDZ. Fifty-four percent of the regenerated shoot rooted on MS + 5 M naphthaleneacetic acid. Histological studies revealed that shoots regenerated via organogenesis.