Changes in the level of endogenous gibberellins and auxins in apical buds ofChenopodium rubrum L. after application of growth substances reversing the effect of (2-chlorethyl)-trimethylammonium chloride (CCC) on flowering

The application of CCC at concentrations inhibiting flowering ofChenopodium rubrum reduces the level of endogenous gibberellins in the apical buds of the plants. The effect of CCC may be reversed by appropriate concentrations of gibberellin (GA-), indole acetic acid (IAA) or kinetin. Kinetin applied to the apical bud during floral induction reduced the level of endogenous gibberellins similarly as CCC and if both CCC and kinetin were applied simultaneously their action was additive. On the other hand IAA applied under the same conditions increased the level of endogenous gibberellins and after joint application of CCC and IAA their level was the same as in untreated control plants. After application of CCC during floral induction the level of endogenous auxins did not change markedly but an active substance “x” appeared on the chromatograms of indole compounds. This substance was found also after simultaneous application of GA- and CCC but not after joint application of CCC and kinetin. If follows from our results that the same morphological phenomenon (flowering) can take place in plants considerably differing as to their level of endogenous growth substances. The ratio of different growth substances is obviously more important than the actual level of the single substances.     

Changes in the level of endogenous cytokinins in apical buds ofChenopodium rubrum L.

CCC (2-chloroethyl)trimethylammonium chloride applied to plants ofChenopodium rubrum during floral induction led to an increase in the level of endogenous cytokinins in the apical buds. Application of gibberellic acid or indole-3-acetic acid at concentrations reversing the effect of CCC reduced the level of cytokinins. After simultaneous treatment with both CCC and one of the growth substances this reduction was less pronounced. From the comparison bf the present results, as well as of those published in previous papers it follows that in apical buds ofChenopodium rubrum there exists a mutual interaction between gibberellins and cytokinins. Under certain conditions both these groups of hormones may substitute for each other in flowering. IAA seems to affect flowering by regulating the level of both gibberellins and cytokinins.     

Changes in the content of endogenous auxins in apical buds ofchenopodium rubrum L. Induced with respect to the endogenous rhythm in capacity to flower

The content of endogenous auxins was examined in apical buds ofChenopodium rubrum plants induced by a photoperiodic cycle of 16h darkness and 8h light followed by a dark period of various duration so as to correspond with either maximal or minimal flowering response in the endogenous rhythm in capacity to flower initiated by the photoperiodic treatment. Apical buds of potentially generative plants contained less auxins than apical buds of plants which remained in the vegetative state. Apical buds from plants treated with kinetin (1. 10^-3 M) and therefore remaining in the vegetative state showed an auxin level comparable to that of untreated plants exhibiting minimal flowering response irrespective of the duration of the second dark period. Plants cultivated on a sucrose solution (0.6 M) during the second dark period became generative even at the normal minimum of flowering. The auxin content of the apical buds was low, similarly as in untreated plants induced for a period leading to maximal flowering response. On the other hand, apical buds from plants grown on sucrose solution during a dark period leading to the manifestation of maximal flowering response showed a relatively high auxin content comparable to that found in untreated plants which had obtained a more extended induction by three photoperiodic cycles. The results are discussed with respect to the possible role of endogenous auxins in the regulation of the changes in growth correlations occurring in the shoot apex during photoperiodic induction and in the expression of the competence to flower.     

Organ correlations and flowering in chenopodium rubrum L.

Correlations within a shoot ofChenopodium rubrum L. ecotype 374 grown under continuous light or photoperiodic flower induction were studied using surgical treatments. Removal of a single pair of shoot organs had a variety of effects depending on position: significant changes in the number of leaf pair on the main axis or in axillary buds and in the height of shoot apices; or no effect on the parameters scored. Flowering was not affected by any of the treatments carried out. Decapitation brought about a significant increase in the number of leaf pairs in axillary buds and flowering was inhibited in 8- and 9-d old plants. Flowering was not affected in 21-d old plants. The role of shoot organ correlations, especially that of apical dominance, in regulation of flowering inC.rubrum is discussed.     

Localization of starch in shoot apices of vegetative and photoperiodically induced plants ofChenopodium rubrutn

Starch was determined by means of IKI reaction in shoot apices ofChenopodium rubrum plants induced to flowering by two short days and in non-induced plants. Small starch grains were already observed in the meristematic cells at an age of four days after sowing. Larger grains were found in the subapical region of the apex. Heterogeneity increases during further growth of the plants in induced, as well as in non-induced vegetative plants. Starch disappears from the cells potentially giving rise to axillary buds, while the number and size of starch grains increase in cells from which leaf primordia will be formed. This metabolic specifity of leaf and bud primordia is preserved during morphological differentiation and applies to vegetative, as well as to prefloral apices of photoperiodically induced plants. The amount of starch in the different regions of the apex is linked rather with organogenesis than with the quantitative growth in the apex.     

Organ correlations inchenopodium rabrum l. shoots studied by Means of32P Distribution

21-day old plants ofChenopodium rubrum L. ecotype 374 were used. Organ relationships in the shoots were investigated by³²P distribution, which indicated different organ correlations in plants grown in continuous light and in plants treated with flower-inducing and non-inducing dark periods. Dark periods were associated with a low³²P distribution in young leaves and a high one in axillary buds. In the following light period the high³²P distribution in axillary buds continued whereas the³²P distribution in the leaves on the main axis increased and was similar to that in plants grown in continuous light. The high³²P distribution in axillary buds was brought about by both, flower-inducing and non-inducing dark treatments. Decapitation resulted in a high³²P distribution in buds, in continuous light an increased³²P distribution was also found in leaves. These effects were not fully cancelled by IAA application. The results are discussed with respect to an assumption that decrease of apical dominance represents a step in a sequence of events leading to flowering.     

Treatments that Enhance Flowering in the Post-inductive Period of a Short-day Plant, Chenopodium rubrum L.

Flowering was promoted in young plants of Chenopodium rubrumL. by application of growth inhibitors such as 5-fluorodeoxyuridine(FUDR) and (2-chloroethyl) trimethylammonium chloride (CCC),growth substances (indol-3yl-acetic acid, IAA), by the removalof roots and by drought. All the treatments were effective onlyduring the post-inductive period and at the threshold levelof photoperiodic induction. The response of plants was strictlytime-dependent. The experimental data indicate that the stimulationof flowering is usually accompanied by inhibition of leaf initiationand growth. The treatments probably produced variation in thequantitative expression of flowering by causing a shift in emphasisin the development of leaf and bud primordia at the shoot apex.The dynamic analysis of differentiation of the shoot apex indicatesa correlation between the morphological stage of the shoot apexand its responsiveness to the treatments.     

Reversal of IAA-induced inhibition of flowering by aminoethoxyvinylglycine inChenopodium

Aminoethoxyvinylglycine (AVG) applied as a droplet (3 μl, 0.1 mM) to the plumule of seedlings of both the short-day plantChenopodium rubrum and the long-day plantChenopodium murale counteracted to a great extent or even canceled the inhibition of flowering due to exogenous indole-3-acetic acid (IAA). This effect was more pronounced with the two substances administered simultaneously than with later application of AVG alone. AVG by itself in some cases promoted the percentage of flowering in bothChenopodium species. Application of IAA to the shoot apex was shown to elevate ethylene production in both species, whereas application of AVG alone was shown to suppress it. Thus, ethylene may be considered an active agent of flowering inhibition brought about by IAA application.     

Action of inhibition by fluorodeoxyuridine and its reversal by thymidine on the nucleic acid fractions of the overground parts of photoperiodically inducedChenopodium rubrum plants

The nucleic acid fractions obtained by chromatography on MAK columns were compared in 4 variants ofChenopodium rubrum plants treated in different ways during floral induction. The first variant was normally induced to flowering. The second one was inhibited by application of FUDR to the apical bud on the third day of induction. In the third variant the inhibitory effect of FUDR was reversed by application of THY 24 h after FUDR treatment. In the fourth variant THY was applied 24 h after the termination of induction at a time when it was no longer able to reverse the inhibition of flowering. In plants treated with FUDR, a decrease in DNA and RNA synthesis was observed. After reversal of the inhibitory effect of FUDR by THY, DNA synthesis remained somewhat lower than in the control variant but RNA synthesis reached the same level as in the controls or even surpassed it. In plants to which THY was applied at a time when it was no longer possible to reverse flowering, the results obtained from different experiments were not identical. In some experiments nucleic acid synthesis remained lower than in the variant in which THY restituted flowering. In other experiments nucleic acid synthesis was fully restituted and reached the same level as in the control variant. This phenomenon is explained by the different size of the experimental plants at the beginning of the different experiments. The results are discussed with respect to the relation of the dynamics of nucleic acids to growth and development of the plants.     

The effect of red light on the level of free IAA

Red light (660 nm) break which cancels the floral inductive effect of the dark period brought about a transitional rise in the level of free IAA in the shoot ofChenopodium rubrum. The higher content of IAA was then found in treated plants at the beginning of the following photoperiod, too. The red light treatment did not change the phase of endogenous rhythm fluctuations of free IAA.     

Brassica campestris as a model for studying the effects of exogenous growth substances on flowering in long-day plants

Photoperiodic responses of seedlings of long-day plantBrassica campestris L. cv. Ceres were investigated at different ages and varying length of inductive period. It was found that photoperiodie response increased with age. All plants flowered after one inductive cycle beginning with a light-period of 16h, but remained in the vegetative phase when kept under short-days (16h darkness, 8h light). Both auxins (IAA and NAA) and cytokinins (kinetin and benzyladenine) inhibited flowering when applied to the plumule or via the roots immediately before the inductive photoperiod. This inhibitory effects was confined to bud formation, whereas the rate of leaf initiation remained mostly unchanged. Only high concentrations of growth substances also affected the growth of roots and leaves. These results agree, in general, with the effects of growth substances in the short-day plantChenopodium rubrum.     

Autoradiography of tritiated uridine in shoot apices ofChenopodium rubrum

The incorporation of uridine-5-³H into shoot apices ofChenopodium rubrum, plants was studied using autoradiography. The evaluation of the rate of incorporation into the nucleolus and the extranucleolar part of the nucleus as a function of the total radioactivity in the apex yields quantitative data on the distribution of labeling in these parts of the nucleus. Incubation of intact germinating plants in uridine-³H makes it possible to carry out chase experiments. Curves of uridine incorporation into the nucleolus and the extranucleolar part of the nucleus were obtained which demonstrated a non-linear course of incorporation. When incubating with uridine from 30 to 120 min the nucleolar/extranucleolar ratio of labelling was found to increase from 2 to 3. In chase experiments this ratio changed within three days from 3 to 1. Interpretation of these results in view of the function of RNA localized in different parts of the nucleus is discussed.     

The incorporation of uridine-3H into the shoot apices of photoperiodically induced and non-induced plants ofChenopodium rubrum L.

The influence of photoperiodic induction on the incorporation of uridine-³H into the shoot apices ofChenopodium rubrum was studied using the technique of autoradiography. No increase in uridine incorporation was detected either during induction lasting three days or immediately after its termination. Pyroninophylia likewise did not rise. However, changes in uridine incorporation related to morphogenetic activity during leaf formation and later during differentiation of inflorescences were well marked. The distribution of label in the nucleus immediately after three inductive cycles shows the ratio of extranucleolar to nucleolar incorporation to be higher in non-induced control plants than in induced ones. Data from literature pointing to an activation of RNA synthesis during transition to flowering are discussed and compared with other systems where ontogenetic changes are accompanied by marked changes in RNA synthesis. It is assumed that the activation of RNA synthesis after induction is connected mainly with the activation of growth. However, inChenopodium rubrum photoperiodic induction proceeds together with limited growth and without activation of RNA synthesis.     

Floral and growth responses in Chenopodium rubrum L. to an extract from flowering Nicotiana tabacum L.

Flowering of Chenopodium rubrum seedling plants was obtained in continuous light after application of fractions of a partially purified extract from leaves of flowering Maryland Mammoth tobacco (Nicotiana tabacum). The stage of flowal differentiation was dependent on the age of the Chenopodium plants used for the bioassay. Apices of plants treated with the extract at the age of four or seven days showed an advanced branching of the meristem or the beginning of formation of a terminal flower; treatment with the extract of plants 12 d old resulted in rapid formation of flower buds in all assay plants. Non-treated control plants kept in continuous light remained fully vegetative. The effects of the extract on flowering were associated with pronounced growth effects. Floral differentiation was preceeded by elongation of the shoot apex. Extension of all axial organs occurred, while growth of leaves, including leaf primordia, was inhibited. The pattern of growth after application of the flower-inducing substance(s) did not resemble the effects of the known phytohormones, but showed some similarities to growth changes resulting from photoperiodic induction of flowering.     

Changes in nucleic acid synthesis in cotyledons and apical buds of chenopodium rubrum l. associated with photoperiodic induction

The nucleic acid (NA) fractions were analyzed in cotyledons and apical buds ofChenopodium rubrum plants by means of acrylamide electrophoresis at the end of the dark period of a different number of photoperiodic cycles or after transfer of the plants to light for 4 h subsequent to the termination of the dark period. The plants were labelled with³²P three hours prior to sampling. The uptake of³²P into the cotyledons was higher in light than in darkness in all cases, however, it was not in correlation with³²P incorporation into the NA fractions. After one dark period lasting 8 or 16 h NA synthesis in light did not increase in comparison with darkness. After two or more photoperiodic cycles NA synthesis was higher in light than in darkness irrespective of whether the dark period lasted 8 or 16 h. NA synthesis was distinctly highest after two inductive cycles lasting 16 h. In buds NA synthesis was slightly shifted in favour of ribosomal RNA as compared with cotyledons. In the cotyledons the increase in light was mainly duo to a raise of rRNA synthesis whereas in the buds synthesis of sRNA and DNA increased, as well.     

Effect of auxins and cytokinins on plant regeneration from hypocotyls and cotyledons in niger (Guizotia abyssinica Cass.)

Tissue cultures were established from hypocotyl and cotyledonary leaf segments ofGuizotia abyssinica Cass. on MS medium supplemented with various concentrations of auxins (IAA, NAA, IBA or 2,4-D) and cytokinins (KN or BA). Expiants cultured on media with cytokinins or in combination with auxins produced shoot buds. Maximum number of shoot buds (20–25 per culture) were differentiated from cotyledonary leaf segments on medium with 2 mg 1^-1 each of KN and IBA. Rooting of regenerated shoot buds was acheived on medium with NAA. The obtained plantlets were successfully transferred to soil.     

Inhibition of flowering ofChenopodium rubrum L. after the action of actinomycin D

Floral differentiation ofChenopodium rubrum is more AD-sensitive than growth of the vegetative organs. With a suitable combination of the manner of application and the concentration of AD used, selective inhibition of flowering can be attained without any effect on growth. The inhibition of flowering was greatest if AD acted during the first two days of photoperiodic induction. With later application its effect on flowering was weaker. RNA synthesized in the first days of photoperiodic induction to a considerable extent ensured its further course.     

Reduction of the Gibberellin Content of Pharbitis Seeds by CCC and After-Effects in the Progeny

Plants of Pharbitis nil were treated with the growth retardant (2-chloroethyl) trimethylammonium chloride (CCC) shortly before and after anthesis. Fresh and dry weight of immature seeds were not affected by the CCC treatment.

The level of gibberellin-like activity in Pharbitis seeds as compared to control seeds was strongly reduced by CCC application. The progenies of the treated plants also had a much reduced GA content in the seedling stage. These results are interpreted to indicate that CCC blocks gibberellin biosynthesis in higher plants, as it does in the fungus Fusarium.

CCC applied via the roots accumulated in the immature seeds and was carried over to the following generation. Consequently, growth of CCC progenies was dwarfed and flower formation inhibited. Both phenomena were overcome by application of gibberellin A₃.

Three gibberellin-like substances (called fractions I, II, and III) were present in Pharbitis seeds and could be separated by thin-layer chromatography. All 3 fractions were also present in seeds treated with CCC. Fractions II and III were present in much higher quantities than fraction I. Both fractions II and III promoted growth of d5 corn but only fraction II was active in dwarf peas grown under red light.

     

Changes of axillary meristem localization in relation to flowering ofRaphanus sativus L. after GA treatment

Two phases of radish ontogenesis (I-when the plant had produced 3 –5 nodes and II-when the plant had produced 8 –10 nodes) were established on the basis of axillary, meristem localization. Flowering of the plants in response to GA treatment depends on the phases in which they were treated and on growth correlations in the apical meristem. The results obtained suggest that the reaction ofRaphanus sativus (LDP) to GA treatment is parallel to that ofChenopodium rubrum (SDP), and that the response of radish plants also depends on changes in growth correlations in the shoot apical meristem at the time of treatment.     

Lipids in plant tissue cultures. VII: Heterotrophic and mixotrophic cell cultures of Chenopodium rubrum

Mixotrophic cell cultures of Chenopodium rubrum were found to synthesize 5 to 33 times more monogalactosyldiacylglycerols and 5 to 16 times more digalactosyldiacylglycerols than heterotrophic ones. The monogalactosyldiacylglycerols and digalactosyldiacylglycerols from mixotrophic cultures contained higher levels of linolenic acid as compared to heterotrophic cultures. It is concluded that the active synthesis of these galactolipids with high levels of constituent linolenic acid is associated with the onset of photosynthesis in plant cell cultures, as is the case in intact plants.