Temporal Morphology and Cap Formation in Acetabularia-Ii. Effects of Morphactin and Auxin

Afin d'apporter des arguments supplémentaires à?hypothese selon laquelle les rhythms circadiens sont impliqués dans la morphogénèse, des expériences ont été réalisées de manière a mettre en évidence la relation éventuelle de dépendance entre le moment (dans le cycle de 24 heures) où commence le traitement et ? effet (1) de la morphactine, un inhibiteur compétitif de ?'auxine et (2) de ? auxine (IAA).

Les résultats ont montré que (1) ? effet inhibiteur de la morphactine varie considérablement selon le moment auquel le traitement a commencé, plusieurs semaines avant ? expression morphogénétique; (2) le maximum d'inhibition change avec le stade de développement et le degré d'inhibition diminue avec le temps; (3) ?'IAA accélerè la formation du chapeau lorsque le traitement a commencé pendant la phase de croissance rapide des algues; ? effet depend du moment (du cycle de 24 heures) auquel il a commencé son effet diminue au cours du temps; (4) lorsque le traitement a commencé avec des algues plus petites que les precédéntes, il exerce un effet transitoirement inhibiteur qui dépend du moment du cycle de 24 heures auquel il a commence; (5) les fragments anucléés aussi répondent différentiellement à un traitement à ? IAA commencé a différents moments du cycle de 24 heures; ? effet est plus net quand des mRNA ont été accumulés; (6) ? effet de ? IAA n'est pas cumulé a celui d'une perturbation du cycle L-D; celui de la morphactine n'est pas modifyé ou est légérement amélioré par une perturbation du cycle L-D.

Mots clefs–Acetabularia, rhymes circadiens, morphogénèse, auxine, morphactine.

In order to support the hypothesis that circadian rhythms are implicated in cap formation, experiments were undertaken on the possible time-dependency of the effects of (a) a competitive inhibitor of auxins, morphactin and (b) of auxin (IAA). It was found that: (i) the inhibitory effect of morphactin varies dramatically with the time at which the several weeks' treatment was first begun; (ii) the maximum inhibition varies with development and decreases with time; (iii) IAA accelerates cap formation when the algae are submitted to IAA during the exponential growth phase; the effect is time dependent and decreases with time; (iv) IAA first applied on smaller algae has a transient inhibitory effect which is time dependent; (v) anucleate fragments also respond differentially to an IAA treatment begun at several times in the 24-hr cycle, most clearly when newly formed mRNA have been accumulated and (vi) the effect of iAA is not cumulative with that of a LD shift; that of morphactin is not, or only slightly, improved by a LD shift.     

Temporal morphology and cap formation in Acetabularia--I. Light-dark cycle perturbations

Cap formation, a major developmental process in the alga Acetabularia, is influenced by a single perturbation of the entraining light-dark schedule and thus, presumably, of the circadian rhythms. This perturbation is brought about several weeks before cap formation, the most conspicuous expression of morphogenesis in Acetabularia. The effect is more pronounced on cap formation than on growth. It varies in importance with the circadian time at which the perturbation was brought about. The effect is dependent on the developmental state of the alga: transfer carried out during the logarithmic phase of growth produces a delay whose importance decreases with time. When carried out during the phase of slow terminal growth, the transfer induces a transitory acceleration of cap formation. When the algae approach their final length, no effect is elicited. Photoperiodism seems to be involved.     

Effect of Morphactin, Gibberellic Acid and Auxin and on Growth and Development of Bryophyllum tubiflorum

six month old LD- and SD- grown plants of Bryophyllum tubiflorum were treated with morphactin (n-buty1-9-hydroxy-fluorene-(9)-carboxylate), singly and in combination with GA₃ and IAA. Morphactin and IAA decreased stem elongation and number of leaves, the effect increasing with concentration concentation. Morphactin also caused lateral buds to develop into branches and the fusion of upper leaves to form structures of different shapes. GA₃ enhanced stem elongation, increased leaf number and induced floral buds under SD conditions. It reversed the inhibitory effect of morphactin on stem elongation, leaf number and leaf fusion and also restored apical dominance when applied simultaneously with morphactin. The stimulaneous application of IAA also reversed the morphactin effects on leaf fusion and on apical dominance. The results have been discussed in the light of literature available on the subject.     

Effect of morphactin on stem growth in relation to auxin in precooled rooted tulip bulbs

Effect of morphactin IT 3456, an auxin transport inhibitor, on tulip stem elongation induced by indole-3-acetic acid (IAA) was investigated. Tulip stem growth induced by IAA 0.1 % in lanolin paste applied on the top internode after excision of flower bud and removal of all leaves was greatly inhibited by 0.2 % morphactin IT 3456 applied on the 4th, 3rd, 2nd and 1st internode. The inhibitory effect of the morphactin on tulips stem growth promoted by IAA was restored by additional application of IAA below the morphactin treatment place. Morphactin inhibited also the growth of all internodes induced by flower bud in the absence of leaves. These results suggest a crucial role of auxin in the control growth of all internodes in tulip stem.     

Plant growth regulators and adventitious root development in relation to auxin

Adventitious root formation in stem cuttings of mung bean was enhanced by ethrel, which had an additive effect when employed simultaneously with indolebutyric acid (IBA). Abscisic acid (ABA) did not influence the number of roots per cutting whereas gibberellic acid (GA₃) and kinetin were without effect on rooting at lower concentrations but were inhibitory at higher concentrations. Nevertheless, all three of these chemicals showed synergistic interactions with IBA and/or indol-3-ylacetic acid (IAA) and thereby significantly promoted root formation. A localised application of morphactin to the epicotyl of cuttings totally inhibited root production irrespective of which of the foregoing growth regulators were suppliedvia the hypocotyl. Morphactin application also prevented root formation in cuttings treated with vitamin D₂. The various growth regulators employed had differing effects on growth of roots but there was no simple relationship between their effects on root formation and subsequent root growth.     

The Effect of Morphactin (Methyl 2-Chloro-9-hydroxyfluorene-9-carboxylate) on Basipetal Transport of Indol-3-ylacetic Acid in Hypocotyl Sections of Phaseolus vulgaris L.

The effect of morphactin (methyl 2-chloro-9-hydroxyfluorene-9-carboxylate)on basipetal transport of auxin (Indol-3-ylacetic acid-2-¹⁴C)was studied in bean (Phaseolus vulgaris) hypocotyl with thedonor-receiver block method. Morphactin (5 x 10^–6m) reduced IAA (5 x 10^–6m) transportintensity by an average of 83 per cent and auxin transport capacityby 90 per cent, but transport velocity was not affected. Morphactin did not inhibit uptake of IAA into hypocotyl tissue,but it did prevent transfer of IAA from the tissue into receiverblocks. Chromatographic analysis of the tissue after 4 h IAA-2-¹⁴Ctransport showed that 54 per cent of the total activity wasin the form of IAA in the control and 42 per cent in the morphactintreated tissue. No difference was found in the rate of decarboxylationof IAA-1-¹⁴C between control and morphactin treated tissue sections.Nor could any difference between control and morphactin be shownin the radioactivity associated with a TCA ppt fraction. Ina study of the transportable auxin pool, morphactin decreasedthe size of the pool and increased the half-life of decay ofauxin transport from 1•22 h to 3•85 h. In a kineticanalysis of the reversal of morphactin (5 x 10^–6m) inhibitionby increasing concentration of IAA-2-14C (5 x 10^–6m to2 x 10^–5m), it was shown that IAA transport resemblesMichaelis-Menten enzyme reaction kinetics, and that inhibitionby morphactin fitted a ‘mixed type’ model. IAA hada dissociation constant of 8•5 x 10^–6m and morphactinthat of 4•3 x 10^–7m with a K_m for the transport processof 8•5 x 10^–6m.     

Temporal Morphology and Cap Formation in Acetabulars-I. Light-Dark Cycle Perturbations

La formation du chapeau, qui constitue un événement morphogénétique de la plus grande importance chez ?'algue Acetabularïa, est influénce par une perturbation unique du cycle lumière-obscurité, done aussi, vraisemblablement, des rythmes circadiens. La perturbation a lieu plusieurs semaines avant ?expression morphogénétique. ? effet est plus marqué sur la formation du chapeau que sur la croissance. Son importance dépend du moment du nyctémère où la perturbation intervient.

?effet dépend du stade de développement de ?'algue: lorsque le transfert d' un régime L-D à un autre a lieu pendant la phase de grande croissance, la formation du chapeau est retardée, mais ? importance de ? effet s'atténue avec le temps. Lorsque le transfert a lieu pendant la phase terminale de croissance (lente), la formation du chapeau est accélérée de façon transitoire. Lorsqu'il a lieu alors que les algues ont presque atteint leur taille finale, il est sans effet. Ces résultats présentent des analogies avec le photopériodisme.

Mots clefs–Acetabularia, rythmes circadiens, morphogénèse, photopériodisme.

Cap formation, a major developmental process in the alga Acetabularia, is influenced by a single perturbation of the entraining light-dark schedule and thus, presumably, of the circadian rhythms. This perturbation is brought about several weeks before cap formation, the most conspicuous expression of morphogenesis in Acetabularia. The effect is more pronounced on cap formation than on growth. It varies in importance with the circadian time at which the perturbation was brought about.

The effect is dependent on the developmental state of the alga: transfer carried out during the logarithmic phase of growth produces a delay whose importance decreases with time. When carried out during the phase of slow terminal growth, the transfer induces a transistory acceleration of cap formation. When the algae approach their final length, no effect is elicited. Photoperiodism seems to be involved.     

Changes in the Cyclic AMP Content during Growth and Development of Acetabularia

The 3',5'-adenosine monophosphate (cyclic-AMP) content of the unicellular alga Acetabularia has been examined at various developmental stages. It has been found that very young algae, less than 10 mm in length, have a high cAMP content [more than 7 pmoles per 100 mg wet weight (WW)], but that with the growth of the algae, the cAMP content decreases rapidly, reaching the low level of 0.5–1.0 pmoles per 100 mg WW. The cAMP content remains at this level until cap differentiation, after which an increase in cAMP content accompanies cap enlargement. It has been shown that these results are unlikely to be affected by changes in the cAMP content induced by variations in circadian rhythm.
Treatment with theophylline (2.10⁻³ M), a phosphodiesterase inhibitor, results in an increase in the cAMP content and delays growth and cap formation. Experiments on the effects of theophylline upon the circadian rhythm of oxygen evolution have shown that the continuous presence of theophylline in the culture medium does not induce a phase shift in the rhythm.
The cAMP content of anucleate Acetabularia shows development stage variations parallel to that of the whole algae.     

Changes in the cyclic AMP content during growth and development of Acetabularia.

The 3',5'-adenosine monophosphate (cyclic-AMP) content of the unicellular alga Acetabularia has been examined at various developmental stages. It has been found that very young algae, less than 10mm in length, have a high cAMP content [more than 7 pmoles per 100 mg wet weight (WW)], but that with the growth of the algae, the cAMP content decreases rapidly, reaching the low level of 0.5--1.0 pmoles per 100mg WW. The cAMP content remains at this level until cap differentiation, after which an increase in cAMP content accompanies cap enlargement. It has been shown that these results are unlikely to be affected by changes in the cAMP content induced by variations in circadian rhythm. Treatment with theophylline (2.10(-3) M), a phosphodieterase inhibitor, results in an increase in the cAMP content and delays growth and cap formation. Experiments on the effects of theophylline upon the circadian rhythm of oxygen evolution have shown that the continuous presence of theophylline in the culture medium does not induce a phase shift in the rhythm. The cAMP content of anucleate Acetabularia shows development stage variations parallel to that of the whole algae.     

Morphactin-induced changes in the cytokinin effect on tissue and organ cultures ofNicotiana tabacum

Summary The influence of a morphactin, chlorflurenol-methylester (CFM), on the growth, the morphogenesis and the isoelectric peroxidase pattern was investigated in both callus cultures (two different tissue culture strains) and multiple bud cultures ofNicotiana tabacum var.Wisconsin. CFM (range of concentration between 10^–6g/ml and 10^–4g/ml) was applied singly, or in combination with a cytokinin, benzylaminopurine (BAP), or with an auxin, indoleacetic acid (IAA), or with IAA plus BAP.In general, the callus growth was inhibited under the influence of CFM. In some of the experiments carried out in hormone-free media, growth stimulation was observed. Even minimal inhibition or stimulation of the callus growth was always accompanied by characteristic changes in the peroxidase patterns.The following results show the influence of the morphactin CFM on cytokinin effects (endogenous cytokinin or equally the exogenously applied cytokinin, BAP). (1) In the multiple bud cultures, BAP and CFM (both substances combined with IAA) similarly caused inhibition of root formation and stimulation of bud formation. The bands in the peroxidase patterns, characteristic of cytokinin action, were accentuated also of those bud cultures which had been treated with BAP or with CFM. (2) In the callus cultures, the cytokinin characteristics appeared under CFM influence in the peroxidase patterns of one of the tissue culture strains only when CFM was applied in combination with BAP and not in combinations of CFM with IAA.The observed morphactin-induced increase in the cytokinin effects could occur via changes in the hormone level of the tissue.     

Effect of morphactin on certain plant growth substances in bean roots

The effect of morphactin (methyl-2-chloro-9-hydroxyfluorene-9-carboxylate) on the content of several plant growth substances in bean roots was determined. Beans (Phaseolus vulgaris L. cv. Spartan) were soaked in aqueous solutions of morphactin, 1 x 10^-4, 1 x 10^-5, and 1 x 10^-6M and grown in moist vermiculite. As controls were used beans grown in water-moistened vermiculite either intact or having the root tips removed (decapped). The roots, morphactin-treated, controls, and the decapped ones were analyzed for indol-3-yl acetic acid (IAA), indol-3-yl acrylic acid (IAcA), indol-3-yl pyruvic acid (IPyA), indol-3-yl lactic acid (1LA), abscisic acid (ABA), gibberellins GA₁, GA₃, GA₄, and GA₉ using gas-liquid chromatographic methods. Morphactin, while affecting the geotropical responses, changed also the growth substance content of roots. IAA, ABA, GA₁, and GA₉ contents decreased, IPyA, IAeA, GA₃, and GA₄ contents were not affected and ILA content increased slightly with increasing dosages of morphactin. Growth substance pattern of decapped roots resembled that of the roots treated with the highest dose, 1 x 10^-4M, of morphactin.     

Effect of auxin on acropetal auxin transport in roots of corn

Acropetal [¹⁴C]indoleacetic acid (IAA) transport was investigated in roots of corn. At least 40 to 50% of this movement is dependent on activities in the root apex. Selective excision of various populations of cells comprising the root apex, e.g. the root cap, quiescent center, or proximal meristem show that the proximal meristem is the critical region in the apex with regard to influencing IAA movement. The quiescent center has no influence and the root cap has only a minor effect. Excision and replacement of the proximal meristem with an exogenous supply of 10⁻⁸ to 10⁻⁹ molar IAA prevents the reduction in acropetal IAA transport which would normally occur in the absence of this meristem. Substituting 10⁻⁹ molar IAA for the excised root cap brings about a significant increase in the amount of IAA moved acropetally, as compared to intact roots with the root cap still in place. From this and previous work, it is concluded that IAA synthesis occurring in the proximal meristem stimulates the movement of IAA from the basal to apical end of the root.     

Does Ethylene Mediate Root Growth Inhibition by Indole-3-Acetic Acid?

The effects of ethylene and of indole-3-acetic acid (IAA) on growth of excised pea root sections have been compared under a variety of conditions. After 16 hours treatment the inhibitory action of IAA is fully reversible on transfer of the root sections to IAA-free solutions. In contrast, inhibition by ethylene is almost totally irreversible. IAA inhibits growth from zero time; ethylene is generally without effect during the first 3 to 6 hours. The inhibitory action of ethylene is dependent on factors such as tissue age and solution composition which have no major effect on IAA inhibition. Ethylene production is enhanced by 100 μm IAA, but conditions which reduce the rate of ethylene evolution 2 to 3-fold at the same IAA concentration fail to affect the inhibitory action of IAA on elongation. It seems unlikely that ethylene can play more than a minor role in mediating inhibition of pea root growth by IAA.     

Morphactin-gibberellin interaction in lettuce seed germination and seedling growth

Morphactin-butylester (a flourene-9-carboxylic acid derivative) inhibited seed germination of two strains of lettuce. Morphactin induced inhibition of germination could be partially or wholly reversed by simultaneous addition of gibberellic acid. However, gibberellic acid played very little part in reversing the inhibitory effect of morphactin on seedling growth. It is concluded that gibberellin can not reverse all the growth effects induced by morphactin.     

Indolyl-3-acetic acid formation byAzotobacter chroococcum

Summary Small amounts of indolyl-3-acetic acid (IAA) were detected in aerated cultures ofAzotobacter chroococcum grown with or withoutl-tryptophane in the medium, but IAA was detected in agar cultures only whenl-tryptophane was present. Most IAA was found in 7-day-old cultures and less in older cultures. Washed cells did not convert tryptophane enzymically to IAA. The time course of IAA formation byA. chroococcum strain A6 has been described and the effect of adding tryptophane to the medium has been studied. In contrast to results elsewhere strain A6 produced traces of IAA in aerated cultures with or without added tryptophane. IAA was detected only after the end of exponential growth when cells had begun to autolyse. The amount of IAA declined as cultures aged. The slight effect ofl- but not ofd-tryptophane in promoting IAA formation in ageing cultures suggests some kind of biological transformation but it seems unlikely that IAA formation is part of the normal metabolic processes of intact Azotobacter cells.     

Sex-related adaptive responses of Populus cathayana to photoperiod transitions

Populus cathayana Rehd., a dioecious tree species, occupies a wide range of habitats in southwest China. Both males and females were grown under two regimes of photoperiod, from mid-length to short-day photoperiod (SD shift), or to long-day photoperiod (LD shift). SD shift triggered leaf senescence in both males and females by decreasing net photosynthesis rate ( A ), transpiration ( E ), and chlorophyll pigment ( Chl ), non-structural carbohydrate (NSC) and indoleacetic acid (IAA) contents, while increasing abscisic acid (ABA), malonaldehyde (MDA) and free proline (Pro) contents. The antioxidant enzyme (e.g. POD, CAT and SOD) activities and capability to maintain ultrastructural integrity also decreased under SD shift. Males exhibited faster leaf senescence than did females, as shown by greater decreases in A , E , Chl and IAA. However, males maintained a less senescent stage than did females, as indicated by higher values of A , Chl , NSC, IAA and antioxidant enzyme activities. Conversely, A , E , NSC and IAA contents and antioxidant enzyme activities were enhanced by lower O₂^•− in females, whereas reduced by higher O₂^•− in males under LD shift. Such sex-dependent responses of P. cathayana to photoperiod transitions showed that males and females possess different adaptabilities, which may relate to sex-specific leaf senescence speed under changing environments.     

农垦58s育性转换与吲哚乙酸氧化酶和过氧化物酶的关系

本文报道了农垦58s和农垦58幼穗发育期长短日下吲哚乙酸氧化酶和过氧化物酶的活性变化。结果表明:58s LD叶片中吲哚乙酸氧化酶活性从花粉母细胞形成期至花粉内容物充实期显著低于58s SD,与游离IAA的积累密切相关,可能影响花粉育性;但过氧化物酶的活性在上述时期显著高于58s SD,与游离IAA的积累无关,其作用尚不清楚。58s LD幼穗及花药中两酶活性在减数分裂期和花粉内容物充实期均较58s SD高,与同期幼穗及花药中游离IAA的亏缺有一定联系,从而可能在一定程度上影响花粉育性。对照农垦58叶片和幼穗及花药中两酶活性变化都不呈现上述现象。     

Cytological study of inhibitory effects of IAA on the root growth in the Pinus silvestris

It has been found that IAA at the concentrations 0.1--10.0 ppm retards the pine root growth and decreases mitotic activity. All the applied concentrations of this hormone cause a decrease of the 3H-thymidine incorporation index and inhibit endomitotic polyploidization in suprameristematic segments. The mean time of the cell cycle prolongs and the heterogeneity of cellular populations increases in parallel with the increase of IAA concentration. The template activity of DNA decreases under the influence of the applied concentrations of IAA; this effect is being particularly strong in the meristematic root segment. IAA exerts also an inhibitory influence on protein synthesis, especially reducing the synthesis of histones.     

Characteristics of the circadian activity rhythm in common marmosets (Callithrix j. jacchus)

The circadian activity rhythm of the common marmoset, Callithrix j. jacchus was investigated by long-term recording of the locomotor activity of 15 individuals (5 males, 10 females) from 1.5 to 8 years old, both under constant illumination and under LD 12:12. The mean period of the spontaneous circadian rhythm was 23.2 ± 0.3 h. Neither sex-specific differences nor a systematic influence of light intensity on the spontaneous period were observed, but the period was dependent on the duration of the trial and on the age of the individual. Due to the short spontaneous period, in LD 12:12 there was a distinct advance of the activity phase with respect to the light time and a masking of the true onset of activity by the inhibitory direct effect of low light intensity during the dark time. After an 8 h delay shift of the LD 12:12, re-entrainment of the circadian activity rhythm required an average of 6.8 ± 0.7 days; the average re-entrainment time after an 8 h phase advance of the LD cycle was 8.6 ± 1.3 day. This directional effect is ascribed to characteristics of the phase-response curve. No ultradian components were observed, either in the LD-entrained or the free-running circadian activity rhythm.     

Dormancy regulation by morphactin in aerial tubers of Begonia evansiana

Summary The sprouting of aerial tubers of Begonia evansiana was promoted by treatment with morphactin. As with cytokinins, the promotion of sprouting occurred in both the immature and mature tubers. Unlike cytokinins, however, morphactin did not stimulate tuber enlargement. The sprout-inhibiting action of applied gibberellin (GA) was overcome by morphactin. The possible mechanism of the inhibitory action of GA is diseussed in relation to apical dominance.