Effects of the Introduction of Agrobacterium tumefaciens T-DNA ipt Gene in Nicotiana tabacum L. cv. Petit Havana SR1 Plant Cells

Transformation of tobacco leaf discs with the ‘cytokinin’ipt gene yielded several transgenic callus tissue lines, respectiveto the kind of ipt construction present in the A. tumefacienscointegrates. Those calli containing an active ipt gene wereable to grow hormone-autotrophically and showed an increasedendogenous cytokinin level in comparison with controls. Analysisof endogenous IAA level did not allow any quantitative correlationwith the cytokinin content. However, a minimal level of auxinseems to be necessary to obtain hormone-autotrophic growth.Exogenously supplied NAA significantly reduced the endogenouscytokinin content without modifying growth characteristics. The varying chlorophyll content in the different callus lineselicited the study of the ultrastructure of the plastids. Thecontrols contained small plastids, often filled with starchor accumulated vesicles that did not allow observation of theinternal membrane system. The ‘Pssu-ipt’ line, havinga higher cytokinin content, showed plastids with an internalmembrane system consisting of stroma and grana thylakoids, butthis structure was lost during subculture. Swollen thylakoidsappeared, the amount of starch was reduced and vesicles wereaccumulating. (Received November 15, 1990; Accepted March 4, 1991)     

Eye evolution at high resolution: The neuron as a unit of homology

Based on differences in morphology, photoreceptor-type usage and lens composition it has been proposed that complex eyes have evolved independently many times. The remarkable observation that different eye types rely on a conserved network of genes (including Pax6/eyeless) for their formation has led to the revised proposal that disparate complex eye types have evolved from a shared and simpler prototype. Did this ancestral eye already contain the neural circuitry required for image processing? And what were the evolutionary events that led to the formation of complex visual systems, such as those found in vertebrates and insects? The recent identification of unexpected cell-type homologies between neurons in the vertebrate and Drosophila visual systems has led to two proposed models for the evolution of complex visual systems from a simple prototype. The first, as an extension of the finding that the neurons of the vertebrate retina share homologies with both insect (rhabdomeric) and vertebrate (ciliary) photoreceptor cell types, suggests that the vertebrate retina is a composite structure, made up of neurons that have evolved from two spatially separate ancestral photoreceptor populations. The second model, based largely on the conserved role for the Vsx homeobox genes in photoreceptor-target neuron development, suggests that the last common ancestor of vertebrates and flies already possessed a relatively sophisticated visual system that contained a mixture of rhabdomeric and ciliary photoreceptors as well as their first- and second-order target neurons. The vertebrate retina and fly visual system would have subsequently evolved by elaborating on this ancestral neural circuit. Here we present evidence for these two cell-type homology-based models and discuss their implications.     

Nitrate Uptake and the Induction of Nitrate Reductase Activity in Wheat (Triticum aestivum L.) Seedlings

Nitrate uptake and the subsequent induction of in vivo nitratereductase activity in wheat were studied by investigating aeuploid and certain ditelosomic stocks which exhibited in vivoactivity significantly greater than that of the euploid. Thekinetics of nitrate uptake were investigated, but the high activitiesof the ditelosomics were not caused by increased uptake of nitrate,although ditelo-7B^L exhibited unusual uptake dynamics. Analysisof the induction of nitrate reductase activity revealed a biphasicgeneral pattern, with an initial rapid phase being followedby a slower but longer period of induction. The induction rateover the second period, although responsible for only a minorproportion of the total activity induced, was positively correlatedwith the final nitrate reductase level, unlike the rate overthe first induction period. Several stocks exhibited high inductionrates over one or other of the two phases, while ditelo- 1 A^sshowed an abnormal monophasic induction pattern. At the endof the second period of induction, nitrate reductase activitybecame more or less steady, except for activity fluctuationsassociated with the time of application of induction stimuli.     

An 15N -- 14C Study of the Role of the Leaf in the Nitrogen Nutrition of the Seed of Datura Stramonium L.

¹⁵N-Nitrate feeding via the transpiration stream and simultaneousfeeding of ¹⁴C via photosynthesis to a leaf-fruit system inD. stramonium indicate that glutamine is the prime recipientof photosynthetically reduced nitrogen in the leaf. Analysisof petiole and seed indicates that glutamine supplies the seedwith most of the reduced nitrogen required for amino acid synthesis.Carbon and nitrogen assimilation in the leaf do not appear tobe directly related in that serine and aspartate and not glutaminereceive the heaviest initial ¹⁴C label.     

Somatic polyploidy examined by flow cytometry in Daphnia

Flow cytometry was used to examine the patterns of somatic polyploidyand to determine the haploid genome sizes in Daphnia. The averageproportions of polyploid nuclei among adult animals of D.pulex,D.longispina, D.pulex x D.longispina and D.magna equalled 27,24, 24 and 24%, respectively. Both interclonal and developmentallyregulated variation were observed in the level of somatic polyploidy.Adult animals expressed more extensive polyploidy than did juveniles.The estimates of the haploid genome sizes (C values) of D.pulex,D.longispina, D.pulex x D.longispina and D.magna equalled 0.32,0.27, 0.26 and 0.37 pg, respectively. These are the smallestgenomes reported in crustaceans. Apparently, somatic polyploidycompensates for the loss of genetic material and gives riseto genetic plasticity which may contribute to the considerablelevel of phenotypic plasticity observed in Daphina.     

Studies on the absorption spectra of chlorophyll a in aqueous dispersions of lipids from the photosynthetic membranes

The absorption spectra of chlorophyll a were studied in aqueousdispersions of four major lipid components present in the thylakoidmembranes. Chlorophyll a in aqueous dispersions of uncharged galactolipidsrevealed two absorption bands, at 670 and 745 nm, when the molecularratio of chlorophyll to lipid was higher than 0.2. The latterband may be due to the formation of microcrystals of chlorophylla. Chlorophyll a in aqueous dispersions of negatively chargedlipids revealed a single absorption band at 670 nm. However,chlorophyll a was decomposed during measurement in these lipiddispersions. The absorption spectra of chlorophyll a in aqueous dispersionsof mixture of galactolipid and charged lipid were apparentlysimilar to those of chlorophyll a in the charged lipid dispersion.Chlorophyll a, however, was not decomposed in these aqueousdispersions of lipid mixtures. It is concluded that the presence of both galactolipid and chargedlipid are necessary to reconstruct the state of chlorophylla dissolved in the lipid phase in the thylakoid membranes. The red absorption band of chlorophyll a in the reconstructedsystem composed of chlorophyll a, charged and uncharged lipids,appeared at 670 nm with a half bandwidth of 22 nm. Analysisof the absorption spectrum in the fourth derivative and thecurve-fitting methods indicated that the red band was composedmainly of a single band with a peak at 670–671 nm. ¹ Present address: Department of Biology, College of GeneralEducation, University of Tokyo, Komaba, Meguro-ku, Tokyo 153,Japan. (Received October 13, 1977; )     

Predator-limited population growth of the copepod Pseudocalanus sp.

The impact of predators on population growth of Pseudocalanussp. was investigated in Dabob Bay, Washington. Mortality ofPseudocalanus sp. was determined from stage-specific survivorship,from seasonal changes in mortality rates of adult males andfemales and from incidence of injuries to adult copepods. Theprincipal predators of adult Pseudocalanus were identified asthe predatory copepod Euchaeta elongata, the omnivorous euphausiidEuphausia pacifica and the chaetognath Sagirta elegans. Predatorattack rates - and prey mortality rates - are highly density-dependentand thus sensitive to prey dispersion in the water column, particularlyto layering in the vertical plane. Predation rates by the threeprincipal predators exceeded 100% of the recruitment rate toadult Pseudocalanus sp. beginning in early summer, thus restrictingpopulation growth. Planktivorous fish predation (by adult three-spinestickleback, Gasterosteus aculeatus, and juvenile chum salmon,Oncorhynchus keta) on Pseudocalanus sp. adults was estimatedto be two orders of magnitude lower than consumption rates bypredatory zooplankton, at a deep water station in July. Analysisof seasonal changes in prey ingested by Sagitta elegans revealedthat Pseudocalanus sp. was the major prey item of S. elegansin April (61.0% of prey) and in June (67.0% of prey), thereafterdeclining seasonally in importance. Predation by S. elegansvaried seasonally with changes in chaetognath stage structure,vertical distribution and diapause, not size structure alone.Although chaetognath recruitment and population growth appearto be directly coupled to the abundance of Pseudocalanus sp.,predation by S. elegans has little reciprocal impact on Pseudocalanussp. population growth; hence asymmetries may occur in the interaction of planktonic prey and predators.     

Retroelement insertional polymorphisms, diversity and phylogeography within diploid, D-genome Aegilops tauschii (Triticeae, Poaceae) sub-taxa in Iran

Background and Aims: The diploid goat grass Aegilops tauschii (2n = 2x = 14) is nativeto the Middle East and is the D-genome donor to hexaploid breadwheat. The aim of this study was to measure the diversity ofdifferent subspecies and varieties of wild Ae. tauschii collectedacross the major areas where it grows in Iran and to examinepatterns of diversity related to the taxa and geography. Methods: Inter-retroelement amplified polymorphism (IRAP) markers wereused to analyse the biodiversity of DNA from 57 accessions ofAe. tauschii from northern and central Iran, and two hexaploidwheats. Key Results: Eight IRAP primer combinations amplified a total of 171 distinctDNA fragments between 180 and 3200 bp long from the accessions,of which 169 were polymorphic. On average, about eight fragmentswere amplified with each primer combination, with more bandsbeing amplified from accessions from the north-west of the countrythan from other accessions. Conclusions: The IRAP markers showed high levels of genetic diversity. Analysisof all accessions together did not allow the allocation of individualsto taxa based on morphology, but showed a tendency to put accessionsfrom the north-west apart from others regions. It is speculatedthat this could be due to different activity of retroelementsin the different regions. Within the two taxa with most accessions,there was a range of IRAP genotypes that could be correlatedclosely with geographical origin. This supports suggestionsthat the centre of origin of the species is towards the south-eastof the Caspian Sea. IRAP is an appropriate marker system toevaluate genetic diversity and evolutionary relationships withinthe taxa, but it is too variable to define the taxa themselves,where more slowly evolving morphological, DNA sequence or chromosomalmakers may be more appropriate.     

Structure and Development of Stomata in Some Leptosporangiate Ferns

The structure and development of stomata are described for 17species of leptosporangiate ferns. In these species the maturestomata are anomocytic, diacytic, Pyrrosia(applied), tetracytic,suspended, or floating type. Stomata are present on both surfacesof floating as well as sub merged leaves of Azolla and Marsilea.In a few species twin stomata, arrested development, and persistentstomatal initials are present Floating stomata result from disintegrationof the anticlinal suspending wall in Pleopeltis, and by detachmentand displacement in Azolla pinnata. The development of stomatais haplocheilic, syndetocheilic, or syndetohaplocheilic. InCyathea spinulosa the guard cell nuclei divide amitoticallyand the resulting two daughter nuclei occupy the opposite polesof the guard cells     

The metamorphosis of visual systems in the sea lamprey

The life cycle of the sea lamprey, Petromyzon marinus, includes two metamorphoses. At the end of a period spent as a blind larva, buried in the mud of streams, a first metamorphosis prepares it to migrate downstream to the sea or a lake for its growth phase. Then, following a second metamorphosis, it migrates upstream as a sexually mature adult to spawn and die. The downstream migrants have a visual system based upon rhodopsin and vitamin A₁, whereas that of the upstream migrants is based upon porphyropsin and vitamin A₂. The livers contain vitamin A₁ at all stages. The sea lamprey therefore exhibits a metamorphosis of visual systems, like those observed earlier among amphibia. The presence of porphyropsin in this member of the most primitive living group of vertebrates, as in fishes and amphibia, supports the notion that porphyropsin may have been the primitive vertebrate visual pigment. Its association with fresh water existence throughout this range of organisms also is consistent with the view that the vertebrate stock originated in fresh water. The observation that in the life cycle of the lamprey rhodopsin precedes porphyropsin is not at variance with the idea that porphyropsin is the more primitive pigment, since this change is part of the second metamorphosis, marking the return to the original environment. The observation that in lampreys, fishes, and amphibia, porphyropsin maintains the same general association with fresh water, and rhodopsin with marine and terrestrial habit, suggests that a single genetic mechanism may govern this association throughout this wide span of organisms.     

Neurophysiological Genetics in Drosophila melanogaster

Neurophysiological genetics is the study of the mechanisms bywhich genes control nervous function and behavior. The transductionof genetic information into neural information is studied atthe level of the neuron through genetic and physiological techniques. The neurons responsible for the leg-shaking action specificto a single-gene mutant of Drosophila melanogaster, Hk¹, havebeen located in three pairs of small regions in the thoracicganglion. The activity pattern of these neurons is coded bythe mutant Hk¹ gene. The center for the specifically patternedleg-shaking action is composed of several motor neurons whoseactivity is governed by the pacemaking activity of at leastone interneuron. As it is most likely that the mutant gene isexpressed autonomously in this interneuron, there is a possibilityof investigating ways in which genes may influence the propertiesof neurons. The activity of the mutant neuron was monitoredintracellularly, and the pattern formation mechanism was studied.The amplitude, duration, and periodicity of the pacemaker potentialand the spike initiation site determine the activity patternresulting in the specific leg-shaking action.     

The Fine-Structural Localization of Phosphatases in Neurosecretory Cells Within the Ganglia of Certain Gastropod Snails

The neurosecretory cells in the cerebral ganglia of the snail,Planorbis trivolvis, have been examined for structural detailsand distribution of phosphatase activities. A more preliminarystudy on the localization of phosphatases in the neurosecretorycells of Helix aspersa has also been made. These neurons inboth species contain typical elementary neurosecretory granuleswhich appear to be elaborated or condensed in the Golgi saccules.Immature elementary granules are identifiable as "primary lysosomes"in that they contain acid phosphatase activity as well as nucleosidephosphatases. The mature elementary granules display no phosphataseactivity. Some saccules of the Golgi are also reactive for severalnucleoside phosphatases, including thiamine pyrophosphatase(TPPase). In Planorbis, TPPase is also present in the cisternaeof the endoplasmic reticulum. Larger, membrane bound, electron-densebodies (lipochondria) are found in close spatial associationwith the Golgi region. These also possess acid phosphatase;in addition, they contain nucleoside diphosphatases and (inPlanorbis) adenosine triphosphatase. Their content of acid phosphataseand the features of their fine structure indicate that theyare lysosomes akin to the dense bodies of vertebrate neurones. The significance and implications of these results are considered,as are other details of the neuronal and glial structure inPlanorbis.     

Formation of the ascidian epidermal sensory neurons: insights into the origin of the chordate peripheral nervous system

The vertebrate peripheral nervous system (PNS) originates from neural crest and placodes. While its developmental origin is the object of intense studies, little is known concerning its evolutionary history. To address this question, we analyzed the formation of the larval tail PNS in the ascidian Ciona intestinalis. The tail PNS of Ciona is made of sensory neurons located within the epidermis midlines and extending processes in the overlying tunic median fin. We show that each midline corresponds to a single longitudinal row of epidermal cells and neurons sharing common progenitors. This simple organization is observed throughout the tail epidermis, which is made of only eight single-cell rows, each expressing a specific genetic program. We next demonstrate that the epidermal neurons are specified in two consecutive steps. During cleavage and gastrula stages, the dorsal and ventral midlines are independently induced by FGF9/16/20 and the BMP ligand ADMP, respectively. Subsequently, Delta/Notch–mediated lateral inhibition controls the number of neurons formed within these neurogenic regions. These results provide a comprehensive overview of PNS formation in ascidian and uncover surprising similarities between the fate maps and embryological mechanisms underlying formation of ascidian neurogenic epidermis midlines and the vertebrate median fin.     

Lotus japonicus Nodulates and Fixes Nitrogen with the Broad Host Range Rhizobium sp. NGR234

Lotus japonicus possesses major advantages as a model legumefor the study of plant-microbe interactions. The relative absenceof genetic information on its normal microbial partner (i.e.,Mesorhizobium loti) could limit its utility in research. Herewe show for the first time that the broad host range Rhizobiumstrain NGR234 nodulates and fixes nitrogen in symbiosis withLotus japonicus ecotypes "Gifu" and "Funakura". We demonstratethat bacterial mutants deficient in nodulation or nitrogen fixationpossess the expected phenotype with L.japonicus. Nodulationof L.japonicus was sensitive to nitrate. Vermiculite was anefficient synthetic growth substrate, allowing axenic growthin Magenta jars. The genetic analysis of the Lotus japonicus-Mesorhizobiuminteraction should be accelerated through the use of this well-definedmicrosymbiont. (Received December 5, 1998; Accepted April 26, 1999)     

Uncoupling Properties of the Herbicide N-n-Pentyl-N-Methyl-N'-(3,4-Dichlorophenyl)-Urea

The herbicide D₅ (N-n-pentyl-N-methyl-N'-(3, 4-dichlorophenyl)-ureacan uncouple oxidative phosphorylation in isolated plant mitochondria.This paper confirms that D₅ is an uncoupler that catalyzes thecollapse of the transmembrane potential gradient by inducinga movement of protons across the membrane. However, D₅ is notitself capable of transporting protons. D₅ gives complete uncouplingat 40 µM, a lower concentration than that required foruncoupling by the n-butyl homologue ‘neburon’. Analysisof the shape of the state 4 stimulation curve suggests thatD₅ might act as a dimer in the membrane. Attempts to demonstrate binding of D₅ to a membrane target gaveambiguous results, binding is not evident at 10 °C and 25°C but might occur at 15 °C and 20 °C. The calculatedherbicide concentration in the membrane (40 µM of which4–0 µM is as the dimer) is high and similar to thatof the major phospholipids. The calculated partition coefficientbetween medium and membrane (3.8 x 103) is in agreement withthe lipophilicity of the substituted urea herbicides. In the presence of a substrate, D₃ blocks both influx and effluxcalcium movement through the mitochondrial membrane but in theabsence of substrate, D₅ induces binding of calcium. Bindingrequires Mg⁺⁺ but not K⁺ or phosphate and leads to a releaseof H⁺. Ruthenium Red causes a partial inhibition of bindingbut no other reagent or ionophore tested had any effect. Sincebinding does not occur in turnip mitochondria which are unableto transport Ca⁺⁺ it is concluded that the effect is not directlylinked to the uncoupling action. The mechanism of action of D₅ is discussed and it is concludedthat D₅ probably acts as a dimer and perturbs membrane structure.The site of action is probably the lipid components of the membrane. Key words: Plant mitochondria, Herbicide, Substituted ureas, Calcium, Uncoupling     

Phytochrome-regulated PIL1 derepression is developmentally modulated

We define the photoresponsiveness, during seedling de-etiolation,of PHYTOCHROME-INTERACTING FACTOR 3-LIKE 1 (PIL1), initiallyidentified by microarray analysis as an early-response genethat is robustly repressed by first exposure to light. We showthat PIL1 mRNA abundance declines rapidly, with a half-timeof 15 min, to a new steady-state level, 10-fold below the initialdark level, within 45 min of first exposure to red light. Analysisof phy-null mutants indicates that multiple phytochromes, includingphyA and phyB, impose this repression. Conversely, PIL1 expressionis rapidly derepressed by subsequent far-red irradiation ofpreviously red light-exposed seedlings. However, the magnitudeof this derepression is modulated over time, in a biphasic manner,in response to increasing duration of pre-exposure to continuousred light: (i) an early phase (up to about 6 h) of relativelyrapidly increasing effectiveness of far-red reversal of repression,as declining phyA levels relieve initial very low fluence suppressionof this response; and (ii) a second phase (beyond 6 h) of graduallydeclining effectiveness of far-red reversal, to only 20% ofmaximal derepression, within 36 h of continuous red light exposure,with no evidence of circadian modulation of this responsiveness,an observation in striking contrast to a previous report forentrained, green seedlings exposed to vegetative shade. Thesedata, together with analysis of phytochrome signaling mutantsand overexpressors with aberrant de-etiolation phenotypes, suggestthat the second-phase decline in robustness of PIL1 derepressionis an indirect consequence of the global developmental transitionfrom the etiolated to the de-etiolated state, and that circadiancoupling of derepression requires entrainment.     

Habituation in Nicotiana bigelovii tissue cultures: Different behaviour of two varieties

The exceptionally high capacity for transformation to autotrophyfor hormones (habituation) discovered in Nicotiana bigeloviiled us to a comparison of the responses of 2 varieties (quadrivalvisand bigelovii) to different hormone treatments applied to theirseeds. Hormones used were: kinetin (0.8, 1.6, 3.2 ppm), 2,4-D(0.1, 0.4 ppm), IAA (2 ppm) and NAA (1, 2, 4 ppm). Callus formation and habituation were scored after transferon minimal medium (without hormones) 3 months after sowing.Var. quadrivalvis showed a much higher ability to form callusbut the callus formed was autonomous in a lower percentage ofcases when IAA or kinetin treatment was considered. Analysisof auxin-like substances in leaves of the 2 varieties showeda higher content in N. bigelovii var. quadrivalvis. Data arediscussed with particular regard to their relevance to tumorousphenomena in plants. ¹ Publication No 52 of the Laboratorio di Mutagenesi e Differenziamento,C. N. R. (Received March 30, 1971; )