Spermidine and flower-bud differentiation in thin-layer explants of tobacco

Three lines of evidence indicate a connection between high spermidine levels and floral initiation in thin-layer tissue cultures of Wisconsin-38 tobacco (Nicotiana tabacum L.). (1) Spermidine levels are much higher in floral buds than in vegetative buds. (2) Inhibition of spermidine synthesis by cyclohexylamine prevents the rise in spermidine titer, inhibits floral initiation and promotes the formation of vegetative buds instead. (3) Application of exogenous spermidine causes floral initiation in cultures which would otherwise form vegetative buds.     

The dose of 1-naphthaleneacetic acid determines flower-bud regeneration in tobacco explants at a large range of concentrations

Short-term applications of very high concentrations of 1-naphthaleneacetic acid (NAA) to expiants from flower stalks of tobacco (Nicotiana tabacum L. cv. Samsun) induced flower-bud regeneration to the same extent as longer or continuous incubation on lower concentrations. The maximum number of flower buds per explant after 15 d of culture was obtained not only by continuous culturing at 1 mol·l^–1 NAA but also by 12 h of culturing at 22 mol·l^–1 or 0.5 h at 220 mol· l^–1, followed by incubation on medium without auxin for the remaining period. Continuous application of such high concentrations resulted in callus formation or caused the death of the explanted tissue. In all experiments in which auxin concentration and time of application were independently varied, the product of concentration and time determined the number of buds formed. Most, but not all, of the NAA taken up by the tissues was converted into conjugates. In expiants which had received a dose which was optimal for regeneration, the internal concentration of free NAA remaining beyond the pulse period was between 1.7 and 6.2 mol·l^–1. Suboptimal applications led to lower values, supraoptimal treatments to much higher internal concentrations. The physiological effect, which depends on the internal hormone concentration, thus manifested itself as dose-dependent with regard to applied hormone.Abbreviations BAP N⁶-benzylaminopurine - NAA 1-naphthaleneacetic acid     

Comparison of de-novo flower-bud formation in a photoperiodic and a day-neutral tobacco

Regeneration of flower buds in thin tissue layers from pedicels of photoinduced short-day (SD) tobacco, Nicotiana tabacum L. cv. Maryland Mammoth, is described. Up to seven flower buds per explant were obtained in a medium containing Murashige and Skoog's macro- and microclements, 100 mg/l myoinositol, 0.1 mg/l thiamine-HCl, 6% glucose, 5 M N⁶-benzylaminopurine, and 0.5 M -naphthaleneacetic acid. Usually some vegetative buds were also formed in the pedicel thin tissue layers. Thin tissue layers from other positions in the induced SD tobacco regenerated vegetative buds only. A comparative study with a day-neutral (DN) tobacco, Samsun, showed that the capacity to form de-novo flower buds was more localized and less strongly determined in photoperiodic than in the DN tobacco. The differences between the photoperiodic and DN tobaccos in flower-bud regeneration capacity are thus quantitative and not qualitative. The basis for this quantitative difference is not known, but may depend on factors controlling production of floral stimulus (florigen) and competency of cells to respond to florigen, and-or stability of the determined state to form flower buds in vitro.Abbreviations BAP N⁶-benzylaminopurine - DN day-neutral - GA₃ gibberellic acid - LD long-day - MM Maryland Mammoth - NAA -naphthaleneacetic acid - SD short-day     

Identification of somatic hybrids in plant protoplast fusions with monoclonal antibodies to plasma-membrane antigens

Monoclonal antibodies generated by immunization with a plasma-membrane preparation from suspension-cultured cells of Nicotiana glutinosa L. were used in combination with fluoresceinor rhodamine-labeled goat anti-mouse immunoglobulins to identify heterokaryons in protoplast fusion procedures. Antibody labeling did not inhibit callus formation nor plantlet regeneration. The antibodies are non-invasive and surface labeling provides clear optical discrimination of true heterokaryons from unfused aggregates as well as from parental protoplasts and homokaryons. Labeling is stable throughout fusion and hence by pre-labeling parental protoplast populations the strategy is both versatile and of general applicability.     

Comparison of photosynthetic parameters of an aurea mutant (Su/su) of tobacco and the wild-type by the photoacoustic method

Oxygen evolution and energy storage yields in tobacco (Nicotiana tabacum L.) wild-type (cv. John Williams Broadleaf) and a mutant (Su/su) deficient in chlorophyll were compared using the photoacoustic technique. Oxygen-evolution and energy-storage quantum yields in the mutant were higher when measured in red light (640–690 nm) than green or blue light (540 nm and 440 nm, respectively), indicating that carotenoids in this mutant do not transfer energy efficiently to the photochemical reaction centers. It is suggested that carotenoids may play a role in protecting the photosynthetic apparatus against damage by high energy fluxes. In the wild-type, the oxygenevolution yield did not change drastically throughout the visible spectrum. The mutant had a higher quantum yield of oxygen evolution than the wildtype. Similarly maximum rates obtained from saturation curves for the mutant were more than twice higher per leaf area and about five times higher per chlorophyll, as compared to the wild-type.Abbreviation PS photosystem     

绵枣儿花芽分化的形态解剖学研究

为探明绵枣儿(Barnardia japonica)在哈尔滨地区的年生长节律以及花芽分化进程,该文以从长白山引种至东北林业大学花卉研究所苗圃内的绵枣儿为材料,采用田间观察法研究绵枣儿的年生长节律,并采用石蜡切片法观察其花芽分化各阶段的形态解剖学特征。结果表明:(1)绵枣儿在哈尔滨地区的生长节律大致可以分为四个时期,即花芽分化与发育期、开花期、结实期、休眠期。(2)绵枣儿花芽分化进程可以分为七个阶段,即4月中上旬,由于土壤温度较低,鳞茎仍处于未分化期; 4月下旬进入花序原基分化期; 5月上旬苞片原基分化; 5月下旬为小花原基分化期; 5月末至6月初花被片原基分化; 6月上旬进入雄蕊原基分化期; 6月下旬为雌蕊原基分化期。该研究明确了绵枣儿在哈尔滨地区的年生长节律和花芽分化各阶段的解剖学特性,为园林应用和新品种的选育提供了一定的科学依据。     

Chromatin changes related to dedifferentiation and differentiation in tobacco tissue culture (Nicotiana tabacum L.)

Non-histone chromosomal proteins (NHP) were isolated from different stages of Nicotiana tabacum L. pith dedifferentiation to callus and callus redifferentiation. The NHP were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis on slab gels and analyzed by densitometry. Simultaneous histological changes are reported. In both processes, some high molecular weight protein (HMWP) bands increase drastically in an induction period, previous to cell proliferation, and decrease when cell division declines. Some low molecular weight protein bands, intense in pith tissue, decrease early when callus is forming and increase when cells differentiate. chromatin template activity is high when cells proliferate, coinciding with maximum HMWP-bands intensity.Abbreviations HMWP high molecular weight proteins - IAA indole-3-acetic acid - LMWP low molecular weight proteins - NHP non-histone proteins - TA template activity     

Ethylene-promoted tomato flower abscission and the possible involvement of an inhibitor

The abscission zone in tomato (Lycopersicon esculentum (L.) Mill. flower pedicels is morphologically distinguishable prior to separation and is delineated by an indentation of the epidermis. Exposure of excised pedicels with the flower attached to ethylene results in abscission within 12 h and this can be accelerated by flower removal. Abscission of excised pedicels with the flower removed takes place in the absence of exogenous ethylene but this is delayed by pretreatment with aminoethoxyvinyl glycine, an inhibitor of ethylene biosynthesis. The data presented support the hypothesis that flower tissue is the source of an abscission inhibitor.Abbreviations AVG aminoethoxyvinyl glycine - IAA indole-3-acetic acid     

Rates of corolla growth in tobacco determined with the plastochron index

The growth of vegetative and reproductive shoots of Nicotiana tabacum L. cv. Xanthi is analyzed with the plastochron index to estimate the relationship between corolla growth and time. The plastochron of leaves 9 through 20 declines steadily at each successive node. The flower plastochron increases steadily during the growth of an individual cyme, with the most distal flower to open having the longest plastochron. Variation in the flower plastochron is the result of variation in the rate of flower initiation, not the growth rate of individual flowers. The corolla has an extended phase of approximately constant relative growth in length (between 0.2 · d^–1 and 0.3 · d^–1) until a peak of growth (0.5 · d^–1) 2–3 d before anthesis. Corollas also have periodic peaks and troughs of growth that are low in amplitude (0.1 · d^–1), but persist throughout most of corolla development. The pattern of corolla expansion contrasts strongly with earlier reports of the pattern of tobacco leaf growth.Abbreviations PI plastochron index - PR plastochron ratio - RGR relative growth rate in length The authors thank: Drs. T. Sage and E.G. Williams for the considerable time and space they invested; the members of Dr. R. Wyatt's laboratory for allowing us to use their computer facilities; A. Tull and M. Smith for their care taken in the green-house. This research was supported by U.S. Department of Agriculture grant GAM-89-01056 and by National Science Foundation grant DCB-87-15799.     

Isolation and characterization of mRNAs accumulated during in-vitro flower bud formation

The development of vegetative and generative buds on thin-layer expiants of tobacco (Nicotiana tabacum L. cv. Samsun) has been studied at the level of translatable mRNA to detect changes in the mRNA population during bud initiation and differentiation, and several quantitative differences were found. By differential screening of a cDNA library obtained from flower-bud-regenerating explants we have isolated a group of six cDNA clones representing genes that are preferentially expressed during in-vitro flower bud formation. Nucleotide sequence analysis of one of these cDNAs, pAP8, showed that the most likely open reading frame has some typical characteristics of, and homology with, extensin-like genes. Northern blot analysis and in-situ hybridization suggest a specific role for these extensin-like genes in flower bud initiation on tobacco pedicel explants.     

Determination for inflorescence development is a stable state,separable from determination for flower development in Pisum sativum L. buds

Terminal meristems of Pisum sativum (garden pea) transit from vegetative to inflorescence development, and begin producing floral axillary meristems. Determination for inflorescence development was assessed by culturing excised buds and meristems. The first node of floral initiation (NFI) for bud expiants developing in culture and for adventitious shoots forming on cultured meristems was compared with the NFI of intact control buds. When terminal buds having eight leaf primordia were excised from plants of different ages (i.e., number of unfolded leaves) and cultured on 6-benzylaminopurine and kinetin-supplemented medium, the NFI was a function of the age of the source plant. By age 3, all terminal buds were determined for inflorescence development. Determination occurred at least eight nodes before the first axillary flower was initiated. Thus, the axillary meristems contributing to the inflorescence had not formed at the time the bud was explanted. Similar results were obtained for cultured axillary buds. In addition, meristems excised without leaf primordia from axillary buds three nodes above the cotyledons of age-3 plants gave rise to adventitious buds with an NFI of 8.3 ±0.3 nodes. In contrast seed-derived plants had an NFI of 16.5 ±0.2. Thus cells within the meristem were determined for inflorescence development. These findings indicate that determination for inflorescence development in P. sativum is a stable developmental state, separable from determination for flower development, and occurring prior to initiation of the inflorescence at the level of meristems.     

Chromosomal control of wheat gliadin protein epitopes: analysis with specific monoclonal antibodies

Summary The genetic relationships between small clusters of monomeric alcohol-soluble wheat (Triticum aestivum L.) grain storage proteins (gliadins) were studied using a panel of monoclonal antibodies and immunoblotting, ELISA, and RIA methods. Use of Chinese Spring nullisomic-tetrasomic lines showed that several narrow-specificity antibodies bound specifically to gliadins encoded by genes located on a single chromosome. In at least one case, antibodies bound to genetic blocks of gliadins, indicating that these block members have structural homology. However, often not all gliadins of a block were recognized by an antibody. For broad-specificity antibodies and some narrow-specificity antibodies, structural genes on several chromosomes were important. Studies with several primitive wheat species indicated that, while antibodies usually bound gliadins from the same genome in bread and primitive wheats, antibodies sometimes bound proteins of quite differing mobilities in the two wheat types. Use of antibodies to identify gliadin blocks is simpler than block analysis based on performing crosses, and should be of value in monitoring genotype/end-use quality relationships.     

Uptake,accumulation and metabolism of auxins in tobacco leaf protoplasts

Uptake and metabolism of exogenous naphthalene-1-acetic acid (NAA) and indole-3-acetic acid (IAA) have been studied in tobacco (Nicotiana tabacum L. cv. Xanthi) mesophyll protoplasts. Both auxins entered protoplasts by diffusion under the action of the transmembrane pH gradient without any detectable participation of an influx carrier. Molecules were accumulated by an anion-trapping mechanism and most of them were metabolized within hours, essentially as glucose-ester and amino-acid conjugates. Protoplasts were equipped with a functional auxin-efflux carrier as evidenced by the inhibitory effect of naphthylphtalamic acid on IAA efflux. Basically, similar mechanisms of NAA and IAA uptake occurred in protoplasts. However, the two auxins differed in their levels of accumulation, due to different membrane-transport characteristics, and the nature of the metabolites produced. This shows the need to estimate the accumulation and the metabolism of auxins when analyzing their effects in a given cell system. The internal auxin concentration could be modulated by changing the transmembrane pH gradient, giving an interesting perspective for discriminating between the effects of intra- and extracellular auxin on physiological processes.Abbreviations BA benzoic acid - C_i/C_e accumulation ratio of auxin - IAAasp N-[3-indolylacetyl]-dl-aspartic acid - NAA naphthalene-1-acetic acid - NAAasp N-[1-naphthylacetyl]-l-aspartic acid - NPA N-1-naphthylphthalamic acid The authors thank Dr. M. Caboche (I.N.R.A, Versailles, France) for his generous gifts of some amide derivatives of 1-NAA, Mr. P. Varennes and Dr. B. Das (I.C.S.N., C.N.R.S., Gif-sur-Yvette, France) for recording and interpreting the mass spectra of NAA glucose ester, and Prof. P. Manigault (Institut des Sciences Végétales, Gif-sur-Yvette) for microscopy measurements of protoplast dimensions. This work was supported by funds from the C.N.R.S, I.N.R.A, and E.E.C.     

Floral-stimulus activity in tobacco stem pieces

The growth patterns of axillary buds of dayneutral tobacco (Nicotiana tabacum L. cv. Wisconsin 38) plants were assessed by using expiants of single buds attached to leafless stem cuttings and allowing the buds to grow to flowering without additional manipulation. Buds located 5, 10 and 15 nodes below the inflorescence were employed. For a given bud position, when a cutting had few internodes the growth pattern of a bud tended to fall into one of two groups: buds that produced few-noded shoots and buds that produced many-noded shoots. For example, in a group of 13 cuttings composed of bud 5 with 2 associated internodes, 11 buds produced 14.2 nodes (range, 11–17) and 2 buds produced 32.0 nodes (range, 30–34). As the number of internodes on the cutting increased, the number of buds producing few-noded shoots increased and the number of nodes produced decreased (e.g. in contrast to the data above, all 5th buds with 6 internodes produced 12.8 nodes; range 11–15). When cuttings from the 3 positions had the same number of internodes, the more apical cuttings had buds that produced fewer nodes (e.g. for cuttings with 6 internodes all 5th buds produced 12.8 nodes, all 10th buds produced 15.5 nodes and 85% of 15th buds produced few-noded shoots with 19.3 nodes). The number of nodes produced by a bud was a function of the original position of the stem piece and not the original position of the bud. That is, bud 5 associated with the 6 internodes below it produced 12.8 nodes and bud 10 associated with essentially the same 6 internodes (i.e. the 6 above it) produced 12.9 nodes while bud 10 associated with the 6 internodes below it produced 15.5 nodes. Thus, the number of nodes produced by a bud was dependent upon the original main-axis position of the cutting as well as the number of internodes on the cutting. Buds forced to grow out in situ on main axes devoid of leaves produced substantially more nodes than similar buds on cuttings. Buds isolated without associated internodes produced many-noded plants with a number of nodes similar to that of plants grown from seed. The simplest interpretation of these data is that stem pieces contain floral-stimulus activity and that this activity is present in a gradient with the highest activity being located in the apical part of the stem.We thank Susan Smith and Harry Roy (Rensselaer) for comments, and the National Science Foundation for financial support (IBN-9003739 to C.N.M.).     

Evolution of flower shape inVeroniceae (Scrophulariaceae)

Floral evolution in the tribeVeroniceae was examined using phylogenetic analysis combining 24 adult morphology and chromosome number characters with 22 qualitative and quantitative floral development characters. Taxa sampled included nine species ofVeroniceae and as an outgroup one species each ofDigitaleae andVerbasceae. Veronica, Besseya, andSynthyris formed one clade, subtended byPseudolysimachion and then by theHebe group;Veronicastrum orWulfenia represent the basal-most branch of the tribe. The ancestral flowers of theVeroniceae may have been small with moderately short corolla tubes and lobes; long corolla tubes arose four times in the tribe and large corolla lobes twice.     

Further experiments on spermidine-mediated floral-bud formation in thin-layer explants of Wisconsin 38 tobacco

We studied the effects of various polyamines on bud regeneration in thin-layer tissue explants of vegetative and floweringNicotiana tabacum L. cv. Wisconsin 38, in which application of exogenous spermidine (Spd) to vegetative cultures causes the initiation and development of some flower buds (Kaur-Sawhney et al. 1988 Planta173, 282). We now show that this effect is dependent on the time and duration of application, Spd being required from the start of the cultures for about three weeks. Neither putrescine nor spermine is effective in the concentration range tested. Spermidine cannot replace kinetin (N⁶-furfurylaminopurine) in cultures at the time of floral bud formation, but once the buds are initiated in the presence of kinetin, addition of Spd to the medium greatly increases the number of floral buds that develop into normal flowers. Addition of Spd to similar cultures derived from young, non-flowering plants did not cause the appearance of floral buds but rather induced a profusion of vegetative buds. These results indicate a morphogenetic role of Spd in bud differentiation. Dedicated to Professor Hans Mohr on the occasion of his 60th birthday     

Altered feedback sensitivity of acetohydroxyacid synthase from valine-resistant mutants of tobacco (Nicotiana tabacum L.)

Acetohydroxyacid synthase (EC 4.1.3.18) has been extracted from leaves of three valine-resistant (Val^r) tobacco (Nicotiana tabacum) mutants, and compared with the enzyme from the wild-type. The enzyme from all three mutants is appreciably less sensitive to inhibition by leucine and valine than the wild-type. Two of the mutants, Val^r-1 and Val^r-6, have very similar enzymes, which under all conditions are inhibited by less than half that found for the wild-type. The other mutant, Val^r-7, has an enzyme that only displays appreciably different characteristics from the wild-type at high pyruvate or inhibitor concentrations. Enzyme from Val^r-7 also has a higher apparent K_m for pyruvate, threefold greater than the value determined for the wild-type and the other mutants. The sulphonylurea herbicides strongly inhibit the enzyme from all the lines, though the concentrations required for half-maximal inhibition of enzyme from Val^r-1 and Val^r-6 are higher than for Val^r-7 or the wildtype. No evidence has been found for multiple isoforms of acetohydroxyacid synthase, and it is suggested that the valine-resistance of these mutant lines is the result of two different mutations affecting a single enzyme, possibly involving different subunits.     

Discrimination between the red- and far-red-absorbing forms of phytochrome from Avena sativa L. by monoclonal antibodies

A set of rat monoclonal antibodies (ARC MAC 48 to 52 and 54 to 56), raised to phytochrome from dark-grown seedlings of Avena sativa L. was tested for the ability to discriminate between the red-absorbing (Pr) and far-red-absorbing (Pfr) forms of phytochrome by indirect enzyme-linked immunosorbent assay. MAC 50 bound more strongly to Pfr and MAC 49 and 52 showed preferential binding to Pr from extracts of dark-grown Avena seedlings; MAC 50 also bound more strongly to Pfr from brushite-purified phytochrome. The remainder of the monoclonal antibodies and a rabbit polyclonal antiphytochrome preparation did not discriminate between Pr and Pfr. The results provide evidence for conformational changes in defined regions of the phytochrome apoprotein upon photoconversion.Abbreviations ELISA enzyme-linked immunosorbent assay - FR far-red light - McAb monoclonal antibody(ies) - PBS phosphate-buffered saline - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome - R red light - PMSF phenylmethylsulphonylfluoride     

Influence of leaves and roots on meristem development inNicotiana tabacum L. cv. Wisconsin 38

The terminal, apical shoot meristem ofN. tabacum cv. Wisconsin 38 normally differentiates into a flower after producing 30 to 40 nodes. The influence of leaves and roots on the regulation of flowering was evaluated by counting the number of nodes produced after removal of leaves or the induction of adventitious roots. Leaf removal has no effect on the number of nodes produced before flower formation. Root induction significantly increases the number of nodes produced before flower formation. The plant behaves as if it were measuring the number of nodes between the meristem and the roots as a means of regulating meristem conversion from vegetative to floral differentiation.     

Characterization of anti-var2CSA-PfEMP1 cytoadhesion inhibitory mouse monoclonal antibodies

Pregnancy-associated malaria (PAM) is associated with the massive sequestration of erythrocytes infected with CSA-binding parasites in the placenta. Natural protective immunity against PAM is acquired during the course of pregnancies, with the development of anti-PfEMP1 antibodies recognizing placental infected erythrocytes (IEs) from different geographical regions. Mouse monoclonal antibodies (mabs) were raised against Plasmodium falciparum variant surface proteins expressed by CSA-binding parasites. These mabs blocked 0-60% of CSA-binding parasite adhesion and immunoprecipitated a 350 kDa 125I-labeled PfEMP1(CSA). Two var2CSA domains expressed on the surface of CHO cells (DBL5epsilon and DBL6epsilon) were identified as the targets of three of four antibodies inhibiting CSA binding. Two of these antibodies also recognized either DBL2x or DBL3x, suggesting that some epitopes may be common to several var2CSA domains. These mabs also specifically selected CSA-binding IEs and facilitated the purification from IE extracts of the native var2CSA ligand. This purified ligand elicited antibodies in immunized mice inhibiting efficiently IE(CSA) cytoadhesion. Based on our findings, we provide the first demonstration that the parasite var2CSA surface protein can elicit inhibitory antibodies and define here the subunits of the var2CSA ligand suitable for use in vaccine development.