Amyloplast formation in cultured tobacco cells; effects of plant hormones on multiplication, size, and starch content

In BY-2 cultured tobacco cells (Nicotiana tabacum L.), depletion of 2,4-dichlorophenoxyacetic acid (2,4-D) and addition of benzyladenine (BA) caused amyloplast formation, a decrease in cell multiplication, and an increase in cell size. These changes were primarily triggered by the depletion of 2,4-D, and facilitated by the addition of BA. An increase in the starch content of BY-2 cells was always accompanied by a reduction in cell multiplication. However, when hormonal conditions were unsuitable for amyloplast formation, the starch content of the cells did not increase, even if cell multiplication was forcibly terminated by the addition of aphidicolin. This result indicates that the hormonal conditions themselves, and not the decrease in cell multiplication, induce amyloplast formation in BY-2 cultured tobacco cells.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - DAPI 4,6-diamidino-2-phenylindole - DMSO dimethyl sulfoxide - NAA 1-naphthylacetic acid - PMSF phenyl methyl sulfonyl fluoride     

Isolation of tobacco DNA segments with plant promoter activity.

We constructed a promoter probe vector, pGVL120, to isolate plant DNA segments with promoter activity in tobacco. Plant nuclear DNA Sau3A fragments were inserted in front of the npt-II sequence, and a mixture of recombinant plasmids was mobilized to Agrobacterium sp. and used to transform tobacco protoplasts. By kanamycin selection, transformed plant cell lines containing NPT-II T-DNAs were isolated. Eight of these cell lines were regenerated and analyzed for the levels of NPT-II activity in stem, root, midrib, and leaf. These levels demonstrated novel regulation patterns in each isolate. One cell line, T20, was analyzed in detail and found to contain four different T-DNAs. One of the recloned T-DNAs, T20-2, contains an insert of 401 base pairs in front of the NPT-II sequence, and by reintroducing this T-DNA into plant cells we could demonstrate that this insert provides a promoter sequence. The NPT-II enzyme activity under the control of the P20 promoter is especially high in stem and root, but low in leaf and callus, both in the originally isolated T20 plant and in independently isolated transformants with the T20-2 T-DNA.     

Repression of isoperoxidase formation in excised tobacco pith by exogenous, auxin-controlled RNA

Previous work has shown that tobacco pith tissue contains two constitutive isoperoxidases migrating toward the anode at pH 9·0. Within 24 hr of aseptic culture on basal medium, such tissue develops five new isoperoxidases, three cathodic and two anodic. The appearance of the new isoperoxidases involves de novo protein formation; it is inhibited by anaerobic conditions, by such inhibitors as Actinomycin D, and by the plant hormone indole-3-acetic acid (IAA). We now find that phenol RNA extracted from parent pith and injected or vacuum infiltrated into cultured pith explants prevents the appearance of the new isozymes; RNA from cultured pith has no such effect. Hydrolysis with 0·3 N KOH, ribonuclease or proteolytic enzymes partially destroys this activity, while treatment with both ribonuclease and proteolytic enzymes completely destroys it. Fractionation of the RNA indicates that part of the repressor activity is associated with an mRNA-like fraction.     

Production of ethylene by excised segments of plant tissue prior to the effect of wounding

Summary A delay of 20 to 30 min precedes a rapid increase in the production of ethylene when segments are excised from the petioles of tomato plants. Measurements made during this early phase may be better estimates of production by petioles on the intact plant than those made at later times.     

In vitro flower bud formation in tobacco: interaction of hormones

External application of auxin and cytokinin is required for the formation of flower buds on thin-layer tissue explants of Nicotiana tabacum cv Samsun. Interaction between both plant growth regulators during this regenerative process has been demonstrated with respect to speed of flower bud initiation and the number of flower buds formed. Separation in time of the hormone application during culture revealed that the cytokinin benzyladenine plays a key role in flower bud initiation whereas auxin (indoleacetic acid) stimulates in particular the differentiation of flower buds. The uptake of each hormone was proportional to the concentration supplied in the medium, and the uptake of either hormone appeared independently of the presence of the other. Metabolism studies showed the conversion of indoleacetic acid by the tissue to at least 13 metabolites after 24 h of culture. In addition, indoleacetic acid metabolism was demonstrated not to be influenced by the uptake and metabolism of benzyladenine. Taken together the results indicate that the interaction of auxin and cytokinin with respect to in vitro flower bud formation is indirect, i.e. does not take place at the level of hormone uptake or metabolism but at some step in the cascade of processes they initiate.     

Positional information and pattern formation in plant morphogenesis and a mechanism for the involvement of plant hormones.

I discuss the possibility of examining pattern formation and morphogenesis in plants in terms of the concept of positional information. Experiments performed on shoot, floral and root apices are interpreted in terms of the theory presented. A model for floral morphogenesis and the interaction of phyllotaxis and shoot morphogenesis is also presented. Finally, some genetic abnormalities of floral morphogenesis are discussed in terms of the main theme of the study.     

Role of hormones on flower bud formation in thin-layer expiants of tobacco

Flower bud formation was studied in thin-layer tissue expiants of epidermis plus subepidermal cortex from the inflorescence ramifications ofNicotiana tabacum cv. Samsun. With appropriate hormone concentrations of BA and NAA expiants from flowerv and fruitbearing stalks regenerate flower buds only, while those from the internodes of the inflorescence ramifications produce generative as well as vegetative buds. In both types of expiants the number of buds formed depend mainly on the hormone concentrations but, in addition, the age of stalks and internodes from which expiants are taken also affects bud formation. Both ABA and JA inhibit flower bud formation in expiants of flower stalks. JA was shown to particularly inhibit bud initiation.     

Multiplication of Musa from excised stem tips.

Rapidly multiplying cultures were established from excised shoot tips of two dessert banana clones ('Philippine Lacatan' and 'Grande Naine') and two plantain clones ('Pelipita' and 'Saba'). Apices cultured on semi-solid media produced single shoots while apices placed in liquid media produced shoot clusters. Individual shoots were induced to form multiple shoot clusters by longitudinally splitting the shoot through the apex. Shoot multiplication was stimulated maximally by 5 mg l-1 benzylaminopurine. Rooted plants were produced by treating shoots with auxins. Growth rates based on increase in f. wt of the four clones were compared. During a 4-week culture period 'Pelipita' showed a fivefold increase in f. wt while 'Grande Naine', 'Philippine Lacatan' and 'Saba' showed increases exceeding tenfold.     

Differential effects of ethylene on pith peroxidase of intact tobacco plants and excised tissue

Ethylene increases the pith peroxidase activity of intact tobacco plants (Nicotiana tabacum) but not of excised pith, either at atmospheric or reduced pressures. In the intact plant, the increased activity involves augmentation of the two constitutive anodic isoperoxidases. In the excised pith, ethylene strongly represses one injury-induced isoperoxidase, while not markedly affecting other isozymes known to be repressed by auxin. Thus, the previously described auxin-induced repression of peroxidase is not due mainly to auxin-induced ethylene formation.     

Histochemical study of vegetative and floral bud formation in tobacco stem expiants

Primary explants from the inflorescence stem of tobacco and primary explants from the stem of vegetative plants, cultivatedin vitro under the same conditions, display different morphogenetic ability. The former give rise mostly to floral buds, whereas the latter exclusively to vegetative ones. Histological and histochemical analyses of both original andin vitro cultivated explants were made. They showed differences in chlorophyll content and alcohol dehydrogenase (AD) activity of the original explants reflecting their different metabolic status. Bud primordia were initiated in the superficial meristematic layer derived from epidermal tissues. Floral or prefloral apices were characterized by a strong AD activity in all cells of the meristem, while in vegetative apices AD activity was restricted to their uppermost parts. A high rate of procambium differentiation connected with leaf primordia formation was typical of vegetative buds. A higher concentration of glucose (5 %) enhanced cell division in explants, which is also correlated with a higher AD activity. The significance of vascular tissues for differentiation of vegetative buds is discussed. Presented at the International Symposium “Plant Growth Regulators” held on June 18-22, 1984 at Liblice, Czechoslovakia.     

The involvement of photosynthesis in inducing bud formation on excised leaf segments of Heloniopsis orientalis (Liliaceae)

The role of photosynthesis in inducing adventitious bud formationon leaf segments of Heloniopsis orientalis was investigated.The effect of white light reached a maximum at about l25 J?m^–2?sec^–1.White, red, blue and far-red light were effective in inducingbud formation, but green light was not. In darkness, bud formationwas induced if sugar was added to the nutrient medium. The photosyntheticinhibitors DCMU and AT blocked the effect of light. Bud formationwas inhibited in CO₂-free air. The requirement of sucrose forbud formation in darkness could be replaced by citrate. It wasconcluded from these results that light appears to induce budson leaf segments through some processes dependent upon photosynthesis. (Received January 11, 1978; )     

Flower formation in vitro in a quantitative short-day tobacco: interrelation between photoperiod and infructescence development

The flowering response of thin layers excised from branch internodes of Nicotiana tabacum cv. Maryland Catterton (quantitative short-day plant for induction) was studied under three photoperiodic treatments. The explants were excised from inflorescences bearing flowers only, flowers and green fruits, or from infructescences with green fruits only. The aim of the study was to investigate the post-inductive photoperiodic effects on in vitro flower bud formation in a quantitative short-day tobacco and the relation with infructescence development. Short days quantitatively enhanced the flower bud regeneration capacities of explants in all stages of development, both as number of explants induced to produce flowers and as mean number of flowers per explant. There was no significant difference in flower bud formation on explants of the first two stages, which produced much more flowers than those of the third stage. Observations in planta showed that, during the 20 days separating the second stage from the first stage, there was no significant difference in the number of floral buds and flowers present on the inflorescence; however, the branch internodes lengthened, as did the floral buds and flowers. During the 10 days leading to the third stage, the number of capsules did not change significantly, but a high rate of floral abscission occurred. The present results show that in Nicotiana tabacum cv. Maryland Catterton short day quantitatively controls not only the inductive step of the flowering process, but also affects the capacity to regenerate flower buds during the late post-inductive phases. The responsiveness to the photoperiodic signal decreases only when the plant exhibits only fruits.     

In vitro formation and development of floral buds on tobacco stem explants: effects of kinetin and other factors

Stem segments were excised from plants of Wisconsin 38 tobacco (Nicotiana tabacum L.) in three regions differing in their distance below the inflorescence. They were cultured in vitro in 8- or 16-hr days. After 8 weeks, floral and vegetative buds were counted, and extent of floral development was assessed. Kinetin at 10(-5)m inhibited formation and development of floral buds regardless of indoleacetic acid concentration. Supplied at this concentration with adequate auxin, kinetin stimulated vegetative bud formation and may have caused floral bud abortion. Indoleacetic acid (>/= 10(-6)m) inhibited vegetative and floral bud formation when supplied with low kinetin concentration (/= 10(-6)m), it inhibited floral bud formation and stimulated vegetative bud formation. More floral buds were formed in 16-hr days than in 8-hr days. Few formed on explants other than those derived from the region nearest the inflorescence regardless of other treatment.     

Stem-swelling and photosynthate partitioning in stem mustard are regulated by photoperiod and plant hormones

The experiment was conducted to study the relationship between stem-swelling and photoperiod and growth hormones by comparing stem swelling with non-stem-swelling stem mustard (Brassica juncea var. tsatsai) plants about their growth characteristics and levels of endogenous gibberellin and cytokinin under different photoperiods. The results here showed that plant biomass was higher in 12-h photoperiod compared to that in long day (LD) and short day (SD), whereas stem growth was much stronger in LD compared to 12-h photoperiod and SD. Exogenous application of 1.0 mM gibberellic acid (GA₃) accelerated stem elongation in SD, but 8.9 μM benzyladenine (BA) failed. The shape of the swollen stem was also found to be associated with day length: a LD promoted stem elongation, while a 12-h photoperiod made the stem oval swollen. Also, stem was shown to have no sign of swelling in plants in SD with a relatively poor growth. The further studies showed that the largest proportion of ¹⁴C photosynthate was allocated to the swelling stems in stem-swelling plants, but to expanded leaves in non-stem-swelling plants, and endogenous gibberellin A₁ (GA₁) and zeatin + zeatin riboside (ZRs) were higher in LD compared to 12-h photoperiod and SD. These results from this experiment indicate that stem growth and swelling is a physiological process of hormonal control, and the photoperiod possibly exerts its influence by altering the balance between the levels of endogenous gibberellins and cytokinins.     

Response of excised Avena coleoptile segments to red and far-red light

Summary In seeking a simple, red light-promoted straight growth test in which phytochrome assays may be conducted without interference by protochlorophyll, the response of excised Avena coleoptile segments to red and far-red light was re-examined. The elongation of apical (non-decapitated) segments is promoted by a brief exposure to red light, and this effect is almost completely nullified by an immediately subsequent exposure to far-red light. Although growth promotion by red light occurs in distilled water alone, the effect is greater on a medium consisting of 0.02 M phosphate buffer, pH 6.2 to 6.4, with 1 to 2% sucrose. Over the pH range 4.5 to 7.4, dark-growth decreases with increasing pH, but the absolute increment brought about by red light is nearly constant. Elongation appears to be entirely the result of increased cell size.Contrary to previous reports, similar results can be obtained with subapical (decapitated) coleoptile segments, although the absolute magnitude of the response is reduced.Research carried out at Brookhaven National Laboratory under the auspices of the U.S. Atomic Energy Commision.     

The effects of light and cotyledon age on growth and root formation in excised cotyledons of Sinapis alba L.

Summary Root formation on excised mustard cotyledons was found to be determined by cotyledon age at excision and by light. Light treatment after excision enhanced root production in cotyledons excised 6 hours to 6 days after the start of imbibition and was essential for root production in cotyledons older than 6 days at excision. Three or more 12 hour light periods after excision were sufficient to induce root production in almost 100% of 7 day old cotyledons. The interposition of up to 5 days of darkness between excision and subsequent culture in the light did not reduce the final rooting percentage but delayed the onset of rooting by a time equivalent to the length of the dark period. The dry weight values of the cotyledons were directly related to the total light energy received after excision.The age and light effects on rooting may be mediated by their effects on the rate of breakdown and on the total levels of available reserves present in the cotyledons.