Translation initiation factors eIF4E and eIFiso4E are required for polysome formation and regulate plant growth in tobacco

Eukaryotic initiation factor eIF4E plays a pivotal role in translation initiation. As a component of the ternary eIF4F complex, eIF4E interacts with the mRNA cap structure to facilitate recruitment of the 40S ribosomal subunit onto mRNA. Plants contain two distinct cap-binding proteins, eIF4E and eIFiso4E, that assemble into different eIF4F complexes. To study the functional roles of eIF4E and eIFiso4E in tobacco, we isolated two corresponding cDNAs, NteIF4E1 and NteIFiso4E1, and used these to deplete cap-binding protein levels in planta by antisense downregulation. Antibodies raised against recombinant NteIF4E1 detected three distinct cap-binding proteins in tobacco leaf extracts; NteIF4E and two isoforms of NteIFiso4E. The three cap-binding proteins were immuno-detected in all tissues analysed and were coordinately regulated, with peak expression in anthers and pollen. Transgenic tobacco plants showing significant depletion of either NteIF4E or the two NteIFiso4E isoforms displayed normal vegetative development and were fully fertile. Interestingly, NteIFiso4E depletion resulted in a compensatory increase in NteIF4E levels, whereas the down-regulation of NteIF4E did not trigger a reciprocal increase in NteIFiso4E levels. The antisense depletion of both NteIF4E and NteIFiso4E resulted in plants with a semi-dwarf phenotype and an overall reduction in polyribosome loading, demonstrating that both eIF4E and eIFiso4E support translation initiation in planta, which suggests their potential role in the regulation of plant growth.     

Induction of somatic embryogenesis in loblolly pine (Pinus taeda L.)

Summary Several factors that may affect induction of somatic embryogenesis in loblolly pine (Pinus taeda L.) were investigated in 1994 and 1995. Megagametophytes containing immature zygotic embryos were excised from seeds as explants. Potassium chloride, silver nitrate, myo-inositol, coconut water, or polyamine was added to the control media (U.S. patent no. 5,036,007) to determine the effects of each single ingredient or their combinations on the initiation of embryogenic tissue. Supplements of myo-inositol at 22.2 mM resulted in increases in frequencies of cell mass extrusion and proliferation compared with the control media in consecutive years. Addition of silver nitrate showed the potential to promote initiation of embryogenic culture. The combination of 10 mM potassium with 29.4 μM silver nitrate achieved the highest frequencies in both extrusion and proliferation of embryogenic tissue. The combination of silver nitrate at 29.4 μM with addition of myo-inositol at 11.1 or 22.2 mM achieved a higher conversion rate from extrusion to proliferation. Polyamine did not significantly affect the induction of somatic embryogenesis, but coconut water was inhibitory. Published with approval of the Director of Arkansas Agricultural Experimental Station.     

Relationship between utilization of dual translational initiation signals and protein processing in Streptomyces

Summary Two sets of the Shine-Dalgarno sequence and the initiation codon (ATG) for translation of a gene encoding the protein SSI (Streptomyces subtilisin inhibitor) were studied in vivo by site-directed mutagenesis. The result shows that each ATG can function as an initiator of translation in either Streptomyces lividans 66 or Escherichia coli. The choice of initiation codon seems dependent on the host strain and is closely related to the processing mechanism of pre-SSI protein. The upstream ATG is presumed to be utilized preferentially giving two cleavage sites in pre-SSI in S. albogriseolus S-3253, the original SSI producer strain.Abbreviations SD Shine-Dalgarno - SSI Streptomyces subtilisin inhibitor     

Effect of N6-isopentenyladenine concentration on growth initiation in vitro and rooting of bilberry and lingonberry microshoots

Buds and shoot tips of wild bilberry (Vaccinium myrtillus L.) and lingonberry (V. vitis-idaea L.) plants were cultured on a modified MS medium containing N⁶-isopentenyladenine (2iP), 9.8–78.4 μM, in order to study the effect of the 2iP-concentration on the initiation of growth. The experiment was first performed in the autumn and repeated in the spring to determine the influence of season on growth initiation. To optimise rooting, three different rooting treatments were tested for the bilberry and lingonberry microshoots. Shoots were rooted either in vitro with 0.49 μM IBA (indole-3-butyric acid) or ex vitro, incubating microshoots in 2.07 mM KIBA-solution (potassium salt of IBA) before planting, or microshoots were planted directly on peat without exogenous auxin. The best 2iP concentration for the initiation of the growth for bilberry was 49.2 μM and for lingonberry 24.6 μM. It was observed that increasing the 2iP concentration at the growth initiation stage increased the number of brownish explants both in bilberry and in lingonberry microcultures. Spring was a considerably better time than autumn for the initiation of new growth, for both species. The results of the rooting test showed that the KIBA-treatment before planting on peat increases rooting efficiency in both bilberry and lingonberry. This revised version was published online in June 2006 with corrections to the Cover Date.     

Benzyladenine-induced inhibition of flowering in Chenopodium rubrum in vitro is not related to the levels of isoprenoid cytokinins

The effect of 6-benzylaminopurine (BAP) on floweringand on endogenous levels of isoprenoid cytokinins wasinvestigated in explanted terminal shoots of Chenopodium rubrum cultivated in vitro. Themother plants were grown under continuous light andexplants were cut off when the 6th leaf primordiumoriginated at the shoot apex. The explants wereexposed to one dark period of 13 hours inductive forflowering or to continuous light on medium with orwithout BAP (0.05;0.2;0.4 mg.l^-1). Undernon-inductive conditions no flowering was observedeither in the control or after BAP treatment. Afterreceiving one inductive dark period, the controlexplants flowered. However, BAP application either atthe beginning of the inductive dark period and/orduring the following light cultivation inhibitedflowering and stimulated initiation and growth of leafprimordia. In the case of the most efficient BAPconcentration (0.05 mg.l^-1) flowering wasinhibited by 80% and the number of leaf primordia wasincreased by 3. Explantation caused a significantincrease in the total amount of endogenous cytokininsin the explants within first 13 h, provided they werekept in light. When explants were kept in darkness,only a slight increase in cytokinin levels wasobserved. BAP treatment had no influence on the levelsof endogenous cytokinins either in light or indarkness. We may thus conclude, that BAP applicationinhibited flowering of photoperiodically inducedterminal shoot explants and stimulated leaf primordiaformation with no significant effect on changes inlevels of endogenous isoprenoid cytokinins. This maysuggest the direct ability of BAP to regulate morphogenesis.     

Effect of the fungal pathogen Verticillium fungicola on fruiting initiation of its host, Agaricus bisporus

Dry bubble disease caused by the fungal pathogen Verticillium fungicola¹ is responsible for large losses to the mushroom (Agaricus bisporus) industry. The pathogen induces various symptoms on the host, bubbles (undifferentiated spherical masses), bent and/or split stipes (blowout) and spotty caps. Inoculation of A. bisporus crops with isolates of V. fungicola var. fungicola of various degrees of aggressiveness showed that the more aggressive isolates induced higher numbers of bubbles. The production of other symptoms did not vary with the isolate of pathogen. The total weight of the crop (healthy and diseased mushrooms) was not significantly affected by the disease, but inoculation with highly aggressive isolates resulted in a significant increase in the total numbers of mushrooms. Two hypotheses are proposed to explain the effect of the pathogen on fruiting initiation in relation to aggressiveness.     

The Hfr status of Pseudomonas aeruginosa is stabilized by integrative suppression

Summary A temperature-sensitive mutant (dna-11) with the phenotype of a mutant defective in the initiation of DNA replication, was isolated from an Hfr-like FP2 donor of Pseudomonas aeruginosa. Reversion of its temperature-sensitive character was achieved by integrative suppression rather than by backmutation or an additional suppressor mutation. The dna-11 mutant proved to be helpful in stabilizing the Hfr status of the original host.     

Ribosome-binding sites on chloroplastrbcL andpsbA mRNAs and light-induced initiation of D1 translation

Chloroplast ribosome-binding sites were identified on the plastidrbcL andpsbA mRNAs using toeprint analysis. TherbcL translation initiation domain is highly conserved and contains a prokaryotic Shine-Dalgarno (SD) sequence (GGAGG) located 4 to 12 nucleotides upstream of the initiator AUG. Toeprint analysis ofrbcL mRNA associated with plastid polysomes revealed strong toeprint signals 15 nucleotides downstream from the AUG indicating ribosome binding at the translation initiation site.Escherichia coli 30S ribosomes generated similar toeprint signals when mixed withrbcL mRNA in the presence of initiator tRNA. These results indicate that plastid SD sequences are functional in chloroplast translation initiation. ThepsbA initiator region lacks a SD sequence within 12 nucleotides of the initiator AUG. However, toeprint analysis of soluble and membrane polysome-associatedpsbA mRNA revealed ribosomes bound to the initiator region.E. coli 30S ribosomes did not associate with thepsbA translation initiation region.E. coli and chloroplast ribosomes bind to an upstream region which contains a conserved SD-like sequence. Therefore, translation initiation onpsbA mRNA may involve the transient binding of chloroplast ribosomes to this upstream SD-like sequence followed by scanning to localize the initiator AUG. Illumination 8-day-old dark-grown barley seedlings caused an increase in polysome-associatedpsbA mRNA and the abundance of initiation complexes bound topsbA mRNA. These results demonstrate that light modulates D1 translation initiation in plastids of older dark-grown barley seedlings.     

A DNA fragment containing the groE genes can suppress mutations in the Escherichia coli dnaA gene

Summary An 8.2 kb fragment of E. coli chromosomal DNA, when cloned in increased copy number, suppresses the dnaA46 mutation, and an abundant protein of about 68 kd (60 kd when measured by us), encoded by the fragment, is essential for the suppression (Takeda and Hirota 1982). Mapping experiments show that the fragment originates from the 94 min region of the chromosome. It encodes several proteins but only one abundant polypeptide of the correct size, the product of the groEL gene. Suppression by the fragment is allele specific; those mutations which map to the centre of the gene are suppressed. Other initiation mutants including dnaA203, dnaA204, dnaA508, dnaAam, dnaC, dnaP and dnaB252 are not suppressed. Most suppressed strains are cold-sensitive suggesting an interaction between the mutant proteins (or their genes) and the suppressing protein or proteins.     

Tissue culture propagation of Mongolian cherry (<Emphasis Type="Italic">Prunus fruticosa</Emphasis>) and Nanking cherry (<Emphasis Type="Italic">Prunus tomentosa</Emphasis>)

In vitro culture establishment, shoot proliferation and ex vitro rooting responses of Mongolian cherry (Prunus fruticosa L.), and Nanking cherry (Prunus tomentosa L.), were examined using various combinations of growth regulators. Dormant buds, taken during winter months, were used as explants. In both species, Murashige and Skoog Minimal Organic (MSMO) solid medium supplemented with 0.49 M indole-3-butyric acid (IBA) and either 4.44 or 8.88 M 6-benzylaminopurine (BA), was the best for culture initiation, and with 8.88–15.16 M BA for shoot proliferation. Good rooting responses were also obtained with shoots produced on media containing 0.91 M thidiazuron (TDZ). Auxin treatments were required for ex vitro rooting of approximately 20 mm long shoots in peat/perlite (1:1 v/v) mixture, at 25 °C, under mist. The best rooting (79%) was obtained with IBA/NAA (naphthaleneacetic acid) (9.80/2.69 M) combination. A commercial rooting powder, Rootone F, containing IBA/NAA (0.057/0.067%), was also effective (73%). The ex vitro rooted plantlets did not require any additional acclimatization prior to transplanting to the regular greenhouse conditions.     

The effect of soil microflora on the survival of cuttings of Epacris impressa

A study was conducted to test the effect of inoculation with soil containing mycorrhizae propagules and other soil micro organisms on survival of stem cuttings of Epacris impressa. With non-sterile soil present, the cuttings were mycorrhizal, more survived and the survivors were healthier, with more flowers and roots, although root area was not increased. Soil inoculum thus increased the success of propagation of Epacris impressa for conservation purposes and in commercial horticulture.     

Stimulation of root development with cyclodextrins on jojoba shoots in vitro

Summary We studied the effect of α- and β-cyclodextrins on in vitro rooting of jojoba (Simmondsia chinensis (Link) Schn.) shoots. When 0.03–0.5 mM of cyclodextrins were added to modified Murashige and Skoog culture medium containing 0.015 mM of indolebutyric acid as 1-wk pulse treatment, rooting percentage increased by approximately 100% with respect to controls. Addition of cyclodextrins induced earlier rooting. An increase in rooting following treatment with cyclodextrins was also observed in the absence of indolebutyric acid, indicating that both compounds promote rooting per se.     

<Emphasis Type="Italic">compact shoot and leafy head 1</Emphasis>, a mutation affects leaf initiation and developmental transition in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The shoot apical meristem (SAM) produces lateral organs in a regular spacing (phyllotaxy) and at a regular interval (phyllochron) during the vegetative phase. In a Dissociation (Ds) insertion rice population, we identified a mutant, compact shoot and leafy head 1 (csl1), which produced massive number of leaves (∼70) during the vegetative phase. In csl1, the transition from the vegetative to the reproductive phase was delayed by about 2 months under long-day conditions. With a reduced leaf size and severe dwarfism, csl1 failed to produce a normal panicle after the transition to reproductive growth. Instead, it produced a leafy panicle, in which all primary rachis-branches were converted to vegetative shoots. Phenotypically csl1 resembled pla mutants in short plastochron but was more severe in the conversion of the reproductive organs to vegetative organs. In addition, neither the expression nor the coding region of PLA1 or PLA2 was affected in csl1. csl1 is most likely a dominant mutation because no mutant segregant was observed in progeny of 67 siblings of the csl1 mutant. CSL1 may represent a novel gene, which functions downstream of PLA1 and/or PLA2, or alternatively functions in a separate pathway, involved in the regulation of leaf initiation and developmental transition via plant hormones or other mobile signals.     

Effects of red and far-red light on cell division in the shoot apex ofSilene coeli-rosa L.

Summary 28-day-old plant ofSilene coeli-rosa were exposed at 1,700 hours to 5 or 10 minutes red light, 5 or 10 minutes far-red light, red followed by far-red, far-red followed by red or maintained in darkness. Measurements of the proportions of cells with the 2 C and 4 C amounts of DNA in the shoot apex of the plants, sampled at 2,000 hours, showed that far-red light promoted an increase in the G2 proportion whereas red light resulted in an increase in the G1 proportion of the cell cycle, relative to the dark controls. Moreover these changes were red, far-red reversible. All light treatments resulted in increases in the mitotic index in the apex compared with the dark controls, suggesting increases in the growth rate. The data implicate phytochrome in a low energy response and suggest that, in the shoot apex, G1 is shortened markedly following exposure to farred light, whilst G2 is shortened the most following exposure to red light. The results are discussed in relation to flower-initiation.     

Two WD-repeat genes from cotton are functional homologues of the <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis><Emphasis Type="Italic">TRANSPARENT TESTA GLABRA1</Emphasis> (<Emphasis Type="Italic">TTG1</Emphasis>) gene

Cotton fibres are single, highly elongated cells derived from the outer epidermis of ovules, and are developmentally similar to the trichomes of Arabidopsis thaliana. To identify genes involved in the molecular control of cotton fibre initiation, we isolated four putative homologues of the Arabidopsis trichome-associated gene TRANSPARENT TESTA GLABRA1 (TTG1). All four WD-repeat genes are derived from the ancestral D diploid genome of tetraploid cotton and are expressed in many tissues throughout the plant, including ovules and growing fibres. Two of the cotton genes were able to restore trichome formation in ttg1 mutant Arabidopsis plants. Both these genes also complemented the anthocyanin defect in a white-flowered Matthiola incana ttg1 mutant. These results demonstrate parallels in differentiation between trichomes in cotton and Arabidopsis, and indicate that these cotton genes may be functional homologues of AtTTG1.     

Callus initiation and regeneration capacities in Brassica species

In order to know the genetic differences of de- and redifferentiation capacities, seven Brassica species (B. campestris, B. nigra, B. oleracea, B. hirta, B. carinata, B. juncea and B. napus) were cultured in vitro, and their response to the medium supplemented with various combinations of auxin and cytokinin hormones was compared. Important factors for callus initiation were shown to be auxin and species. Calli were induced most frequently in Murashige and Skoog (MS) medium with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), whereas -naphthaleneacetic acid (NAA) induced preferentially roots at a concentration of 2 to 5 mg/l. Callus-, root- and shoot-forming capacities from explanted cotyledon tissues were significantly different among the seven Brassica species. Calli derived from cotyledons and hypocotyls of seven species were transferred to MS media with 20g/l sucrose, 0 to 0.1 mg/l NAA and 0 to 4 mg/l kinetin to compare their regeneration capacities. Among the seven species tested, B. napus (2n=4x=38, genome AACC) had the highest shoot forming capacity (20.0%). Other amphiploid species, B. carinata (2n=4x=34, BBCC) and B. juncea (2n=4x=36, AABB) formed shoots at low frequencies (2.8% and 1.2%, respectively). A diploid species, B. oleracea (2n=2x 18, CC) also showed high shoot formation (10.2% on average). This result suggests that the gene(s) controlling shoot formation may be localized in the C genome. Differences were also found among varieties and cultivars within a species. One of the cultivars, Siberian kale (B. oleracea var. acephala) gave about 50% shoot formation. This kale was shown cytologically to be an autotetraploid (2n=4x=36, CCCC), thus probably possessing a double set of the shoot-forming gene(s).     

Micropropagation of Chokecherry and Pincherry cultivars

In vitro culture establishment, shoot proliferation and ex vitro rooting responses of chokecherry (Prunus virginiana L.), `Garrington', and pincherry (P. pensylvanica L.f), `Mary Liss' and `Jumping Pound', were examined using various combinations of growth regulators. Dormant winter buds were used as explants. MSMO medium supplemented with 0.49 μM IBA and either 4.44 or 8.87 μM BA was found to be optimal for culture initiation of both species and cultivars. GA₃ (28.89 μM) significantly reduced (p=0.0001) the number of successfully established cultures. BA concentrations 8.87–12.82 μM gave optimal shoot proliferation in chokecherry and 4.44 μM BA in both cultivars of pincherry. Auxin treatments were required for ex vitro rooting of approximately 10 mm long shoots in peat/perlite (1:1 v/v) mixture, at 25 °C, under mist. The best rooting (84%) was obtained with IBA/NAA (9.80/2.69 μM). A commercial rooting powder, Rootone F, containing IBA/NAA (0.057/0.067%) mixture, was also effective (75%). The ex vitro rooted plantlets did not require any additional acclimatization prior to transplanting to the regular greenhouse conditions. This revised version was published online in August 2006 with corrections to the Cover Date.     

Enhanced nodule initiation on alfalfa by wild-typeRhizobium meliloti co-inoculated withnod gene mutants and other bacteria

Nodule formation on alfalfa (Medicago sativa L.) roots was determined at different inoculum dosages for wild-typeRhizobium meliloti strain RCR2011 and for various mutant derivatives with altered nodulation behavior. The number of nodules formed on the whole length of the primary roots was essentially constant regardless of initial inoculum dosage or subsequent bacterial multiplication, indicative of homeostatic regulation of total nodule number. In contrast, the number of nodules formed in just the initially susceptible region of these roots was sigmoidally dependent on the number of wild-type bacteria added, increasing rapidly at dosages above 5·10³ bacteria/plant. This behavior indicates the possible existence of a threshold barrier to nodule initiation in the host which the bacteria must overcome. When low dosages of the parent (10³ cells/plant) were co-inoculated with 10⁶ cells/plant of mutants lacking functionalnodA, nodC, nodE, nodF ornodH genes, nodule initiation was increased 10- to 30-fold. Analysis of nodule occupancy indicated that these mutants were able to help the parent (wild-type) strain initiate nodules without themselves occupying the nodules. Co-inoculation withR. trifolii orAgrobacterium tumefaciens cured of its Ti plasmid also markedly stimulated nodule initiation by theR. meliloti parent strain. Introduction of a segment of the symbiotic megaplasmid fromR. meliloti intoA. tumefaciens abolished this stimulation.Bradyrhizobium japonicum and a chromosomal Tn5 nod^- mutant ofR. meliloti did not significantly stimulate nodule initiation when co-inoculated with wild-typeR. meliloti. These results indicate that certainnod gene mutants and members of theRhizobiaceae may produce extracellular signals that supplement the ability of wild-typeR. meliloti cells to induce crucial responses in the host.Abbreviations EH emergent root hairs - kb kilobase - RDU relative distance unit - RT root tip This is journal article No. 188-87 of the Ohio Agricultural Research and Development Center     

The Effect of Rearing Temperature on Flight Initiation of Trichogramma sibericum Sorkina at Ambient Temperatures

Trichogramma sibericum Sorkina was reared in the laboratory at three temperatures: 16, 21, and 26°C. Individuals from each of these treatments were then tested for propensity to initiate flight at one of four ambient temperatures: 16, 19, 21, or 26°C. Both rearing and ambient temperatures had significant effects on flight initiation. Insects reared at 16°C had the highest mean proportion of flyers; insects reared at 26°C had the lowest. The proportion of insects initiating flight increased with increasing ambient temperature. Also, the interaction of these two temperature experiences was significant. Insects reared at 16°C were more likely to initiate flight at 16°C than insects reared at 21 or 26°C. These results indicate that performance (as assessed by flight initiation) at ambient temperature is dependent on the temperature previously experienced during rearing.