Modulation of TDZ-induced morphogenetic responses by anti-auxin TIBA in bud-bearing foliar explants of Kalanchoe pinnata

This investigation was conducted to study the range of TDZ-induced morphogenetic responses displayed by foliar explants of Kalanchoe pinnata with or without an epiphyllous bud and to assess their possible auxin mediation by using potent anti-auxin TIBA. Each epiphyllous bud developed into a healthy plantlet when cultured in vitro without nutrient/hormonal supplements. The bud on 10⁻⁶ M TDZ-treated leaf disc produced a compact, dwarf and rootless plantlet having achlorophyllous leaves with increased number of marginal notches. TDZ also caused a significant enlargement of the leaf disc as well as induced an organized hypertrophic growth at a specific site, with either effect being more pronounced in discs without an epiphyllous bud. Treatment with 10⁻⁴ M TIBA completely checked the plantlet growth. In combined dispensations, the TDZ-induced responses were modified by the anti-auxin TIBA in more than one way. Whereas it caused a complete reversal of hypertrophic growth on leaf discs and partially restored pigmentation of plantlet leaves, it could not annul the leaf disc enlargement. The diverse morphogenetic modulations were tissue and response specific. A complete reversal of TDZ-induced leaf disc hypertrophy by TIBA indicated an auxin mediation, whereas a complete non-reversal of the induced leaf disc enlargement pointed to the non-involvement of auxin, for the respective responses. The latter response could probably be manifested through cytokinin-activity of TDZ, while still others may not be interpreted exclusively through either one of the two hormonal options.     

Regulation of senescence in bean leaf discs by light and chemical growth regulators

The senescence of excised discs of primary leaves of Phaseolus vulgaris, L., var. Red Kidney was followed by measuring the net breakdown of protein and chlorophyll. The chemical growth regulators indoleacetic acid, 2,4-dichlorophenoxy-acetic acid, gibberellic acid, kinetin, and 6-benzylaminopurine were relatively ineffective in retarding senescence in this tissue. White light, on the other hand, was very effective in senescence retardation. The response to light did not have the characteristics of a low energy (phytochrome) response and was blocked by concentrations of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea which inhibited photosynthesis in the leaf discs. The light-induced retardation of senescence was concluded to be dependent on photosynthesis.     

Effects of 6-benzylamino-purine and 1-naphthaleneacetic acid on the epiphyllous bud formation in Bryophyllum

Summary The effects of the kinin 6-benzylamino-purine and of 1-naphthaleneacetic acid (NAA) on the epiphyllous bud formation in Bryophyllum were studied under controlled environment.In B. daigremontianum which requires long days for epiphyllous budding, buds were formed under continuous short days after application of the kinin. Similarly, such a treatment caused budding in attached non-aging leaves of B. calycinum which normally form buds only after detachment from the plant. This stimulatory effect of the kinin was strictly bound to the treated leaves (or leaf parts), which also showed an increased growth compared with the opposite non-treated leaves. Root formation in the developing buds was inhibited by the kinin.In both species NAA inhibited the epiphyllous budding under inductive conditions. A similar inhibitory effect was exerted by terminal and axillary buds.The results are discussed in the light of other investigations in this and related fields. It is concluded that epiphyllous bud formation is under the control of a correlative inhibition similar to apical dominance. It is further concluded that even though day-length controls both flowering and epiphyllous budding in B. daigremontianum the two processes must be affected through different biochemical systems.     

Interrelationship between ethylene and growth regulators in the senescence of lettuce leaf discs

The interrelationship between ethylene and growth regulators in the senescence of romaine lettuce (Lactuca sativa L.) leaves was studied. Gibberellic acid (GA₃), kinetin, and 3-indoleacetic acid (IAA) retarded chlorophyll loss from leaf discs which were floated on hormone solutions. Abscisic acid (ABA) and ethephon enhanced chlorophyll loss and antagonized the senescence-retarding effect of GA₃ and kinetin. A high concentration of IAA (10^–4 M) caused accelerated chlorophyll loss, whereas a similar concentration of kinetin neither retarded nor promoted chlorophyll loss. The ineffectiveness of IAA and kinetin at their supraoptimal concentrations in retarding leaf senescence was related to increased production of ethylene induced in the treated leaf discs. GA₃ was the most effective in retarding chlorophyll loss and did not stimulate ethylene production at all. The senescence-enhancing effect of ABA was not mediated by ethylene. However, the moderately increased production of ethylene, induced by relatively high concentrations of ABA, could act synergistically with the latter to accelerate chlorophyll loss. It is proposed that the effectiveness of exogenously applied hormones, both in enhancing and retarding senescence, is greatly affected by the endogenous ethylene concentration of the treated plant tissue.Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel, No. 2571-E, 1988 series.     

Patterns of ehtylene production in senescing leaves

Changes in the patterns of ethylene production, chlorophyll content, and respiration were studied in relation to the senescence of intact leaves and leaf discs. The primary leaves of pinto bean, which abscise readily during natural senescence, and tobacco and sugar beet leaves, which do not abscise, were used. A decrease in the rate of ethylene production and respiration, during the slow phase of chlorophyll degradation, was observed in leaf-blade discs cut from mature leaves and aged in the dark. During rapid chlorophyll loss both ethylene production and respiration increased and then decreased. These climacteric-like patterns were shown by leaf discs of all three species. Discs taken from leaves that had been senescing on the plant also showed a climacteric-like rise in ethylene production but not in respiration, which decreased continuously with leaf age. Climacteric-like patterns in the rise of ethylene and respiration for leaf discs were also shown by the petioles of both bean and tobacco leaves. This indicates that the rise of ethylene and respiration is characteristic of the general process of senescence in leaves and is not restricted to the abscission process. In contrast to the ethylene-forming systems in climacteric fruits and many flowers, the one in leaves declines sharply in the early stages of senescence. The subsequent rise of ethylene production appears to be associated with the rapid phase of chlorophyll breakdown, and may indicate the final stage of the senescence process during which ethylene could be actively involved in inducing leaf abscission.     

Water relations of cotton plants under nitrogen deficiency

Nitrogen deficiency in cotton plants (Gossypium hirsutum L.) increased the threshold water potentials for both stomatal closure and leaf senescence (defined as loss of chlorophyll and protein) during drought. These studies attempted to answer two questions: (1) What is the basis for the N/water interaction on senescence? (2) Is there a direct relationship between stomatal closure and senescence? Young and old leaves from N-deficient and N-sufficient plants maintained their relative sensitivities to water stress when excised leaf discs were floated on solutions of polyethylene glycol in dim light. In this leaf disc system, both leaf aging and N deficiency increased the threshold water potential for senescence. Leaf aging and N deficiency also decreased the concentration of exogenous abscisic acid necessary to initiate senescence in discs. A role for cytokinins in controlling senescence could not be clearly shown. In young leaves of both N-deficient and N-sufficient plants, stomata closed at water potentials much higher than those causing senescence. During leaf aging, the water potentials causing senescence increased more than those causing stomatal closure. The two processes thus occurred at about the same potentials in the oldest leaves. These data argue against a general cause-and-effect relationship between stomatal closure and senescence. Rather, each process apparently responded independently to absicsic acid accumulated during drought.     

Apple leaf senescence: leaf disc compared to attached leaf

Attached apple leaves (Pyrus malus L., Golden Delicious) began to lose protein in early August as the first sign of senescence. Apple leaf discs prepared from samples before early August gained protein for up to 7 days after detachment. After early August, the loss of protein from leaf discs was no greater than the loss from attached leaves in 7 days. The loss of chlorophyll from leaf discs began over 2 months before attached leaves began to lose chlorophyll naturally and before leaf discs lost protein. Leaf discs from presenescent leaves did not senesce significantly faster when maintained in darkness instead of 12 hours of light. In general, the loss of protein and chlorophyll from apple leaf discs after 7 days was much less than for most other leaf types studied.     

Regulation of monocarpic senescence of Brassica campestris by the developing pods

Senescence of Brassica campestris L. cv. B-9 was studied with regard to seed maturation and source-sink relationships. In normal control plants leaf senescence (as determined by the change in chlorophyll level) started and proceeded in a progressive manner from base to apex during the period of early pod setting. Complete yellowing of the leaves occurred well before the seed maturation and pod wall senescence. The pod wall always senesced before the attainment of final seed weight. In two different sets of acrocarpous plants containing 65 pods and 10 pods, respectively, leaf senescence was delayed during the pod filling period. It started non-sequentially after complete yellowing and senescence of the pod wall. The degree of leaf senescence at the post-pod filling stage was almost proportional to the number of pods present. When peduncles of the acrocarpous 10-podded plants were removed after the pod filling stage of the plant, leaf senescence was delayed compared to plants whose pedicels were removed, although the senescence pattern of the upper three leaves was nonsequential in both cases. Defruiting at an early stage of development delayed leaf senescence, although the pattern of such senescence remained unaltered (i.e. nonsequential). Defoliation hastened the seed-filling process and pod wall senescence. Plants containing fewer pods had higher average seed weight, although yield per plant was reduced.
These results suggest that the pod wall serves as a temporary as well as intermediary storage organ and that foliar senescence is not directly related to seed maturation. The possible cause of uncoupling between foliar senescence and seed development is discussed.     

Effect of wounding on ethylene biosynthesis and senescence of detached spinach leaves

Parameters of senescence and ethylene biosynthesis pathway were screened simultaneously in detached spinach leaves and leaf discs. Senescence was enhanced by application of 1-aminocyclopropane-1-carboxylic acid (ACC) and was retarded by amino-ethoxyvinylglycine (AVG). Evidence is presented showing that the bursts of both wound- and climacteric-like ethylene promoted senescence of detached leaves and leaf discs. This ethylene-enhanced leaf senescence was dependent on: (a) ethylene production rates in the tissue; (b) the degree of wounding. Wounding resulted in elevated levels of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), which declined in advanced stages of senescence. The results suggest that wounding might be regarded as one of the primary events in the induction of the senescence syndrome in detached leaves and leaf discs, while ethylene is implicated as a regulator of the rate of the process.     

ACC synthase expression regulates leaf performance and drought tolerance in maize

Ethylene regulates entry into several types of plant developmental cell death and senescence programs besides mediating plant responses to biotic and abiotic stress. The response of cereals to conditions of drought includes loss of leaf function and premature onset of senescence in older leaves. In this study, ACC synthase ( ACS ) mutants, affecting the first step in ethylene biosynthesis, were isolated in maize and their effect on leaf function examined. Loss of ZmACS6 expression resulted in delayed leaf senescence under normal growth conditions and inhibited drought-induced senescence. Zmacs6 leaves continued to be photosynthetically active under both conditions indicating that leaf function was maintained. The delayed senescence phenotype associated with loss of ZmACS6 expression was complemented by exogenous ACC. Surprisingly, elevated levels of foliar chlorophyll, Rubisco, and soluble protein as well as improved leaf performance was observed for all Zmasc6 leaves, including young and fully expanded leaves which were far from initiating senescence. These observations suggest that ethylene may serve to regulate leaf performance throughout its lifespan as well as to determine the onset of natural senescence and mediate drought-induced senescence.     

Prolonged leaf senescence in Clusia multiflora H.B.K.

 Leaf aging and senescence in Clusia multiflora H.B.K. was investigated by artificial treatments, such as floating leaf discs on water in darkness, or darkening leaves attached to the parent plant in situ in trees living in a tropical cloud forest. In both cases several parameters modified by age were evaluated such as nitrogen levels, chlorophyll content, succulence and carbohydrates levels. A prolonged senescence (nearly 3 months in floating leaf discs) was observed, contrasting with species such as Heliocarpus americanus (5 days) and Cecropia palmatisecta (20 days), characterized by low values of leaf weight per area, but similar to species with high leaf weight per area and with high levels of organic acids such as Clusia minor and Fourcroya humboldtiana, where acids may act as a reserve of C and energy. After 30 days in darkness C. multiflora leaf samples collected in the field did not show differences in comparison to non-darkened opposite leaves with respect to chlorophyll, titratable protons and carbohydrates, and leaves performed photosynthesis after 2 months in darkness. The effect of age in leaves was evaluated in a gradient of leaves, sampled at different positions from the apex and ranging in age from 15 days to 2 years old. The study of senescence in tropical wild plants is uncommon, but it is important knowledge for understanding foliar development, and response to internal rather than environmental regulation in climates where seasons are not strongly marked as is the case in the tropical mountain forest, where C. multiflora constitutes an important component in the early successional vegetation. Received: 21 October 1996 / Accepted: 12 November 1996     

Changes in Photosynthetic Activity and Related Processes Following Decapitation in Mulberry Trees

Physiological responses to decapitation, in combination with bud removal or bud retention, were followed for 45 days in mature leaves of potted mulberry trees (single shoot with 24 to 28 leaves) held in a greenhouse. Mature leaves, whose photosynthetic activity had already attained a maximum, initially increased and subsequently maintained their rates of gas exchange after decapitation. Equivalent leaves on intact trees showed a gradual decline in photosynthesis together with other changes generally associated with early senescence viz. loss of chlorophyll, increased starch, and accumulation of one category of cytokinin-like material presumed to be a glucose ester. Maintenance of physiological activity following decapitation, especially when combined with bud removal, was associated with greater chlorophyll content, mesophyll cell enlargement (palisade cells appeared more elongate), lower starch, and alteration in foliar levels of cytokinin-like substances. Internal constraints on CO₂ assimilation, i.e. residual resistance (rr), rather than stomatal factors, appeared to be the major influence on gas exchange. The higher photosynthetic activity of leaves on decapitated trees relative to control trees of the same age was attributed to lower r, but was also associated with higher chlorophyll content (leaf area basis) so that CO₂ assimilated per unit chlorophyll was not substantially altered by treatment.     

The relation between nitrogen deficiency and leaf senescence

Because the "mobilization" of nitrogen resulting from nutritional nitrogen deficiency is also prominent during leaf senescence, the characteristics of these two syndromes were compared. Oat plants ( Avena sativa L. cv. Victory) were raised on a nutrient solution, complete except for nitrogen supply (i.e., with only the seed protein as nitrogen source), and the senescence of their leaves was compared with that of controls grown on a full nutrient solution. The N-deficient plants flowered after forming only 4 leaves and each set a single seed. The nitrogen lack affected the content of chlorophyll somewhat more than the content of the amino acids or protein nitrogen. However, spraying the plants with kinetin solution was able to retain 20–30% of the chlorophyll and protein. During senescence, the chlorophyll appears to be less stable in the N-deficient leaves than in the controls, while the protein is somewhat more stable than in the controls. Also, when the detached leaves from N-deficient plants senesced in white light or in darkness, kinetin delayed their senescence almost as effectively as that of control leaves. Most strikingly, the stomata of N-deficient leaves after detachment and floating on water were largely closed in light, just as in senescence, but could be partially induced to open by kinetin treatment. Since stomatal closure has earlier been shown to cause senescence, the characteristic syndrome of foliar nitrogen deficiency is concluded to be partly that of senescence.     

Production and action of ethylene in senescing leaf discs: effect of indoleacetic Acid, kinetin, silver ion, and carbon dioxide

Supraoptimal concentrations of indoleacetic acid (IAA) stimulated ethylene production, which in turn appeared to oppose the senescence-retarding effect of IAA in tobacco leaf discs. Kinetin acted synergistically with IAA in stimulating ethylene production, but it inhibited senescence. Silver ion and CO(2), which are believed to block ethylene binding to its receptor sites, delayed senescence in terms of chlorophyll loss and stimulated ethylene production. Both effects of Ag(+) were considerably greater than those of CO(2). IAA, kinetin, CO(2), and Ag(+), combined, acted to increase ethylene production further. Although this combination increased ethylene production about 160-fold over that of the control, it inhibited senescence. Treatment with 25 mul/l of ethylene in the presence of IAA enhanced chlorophyll loss in leaf discs and inhibited by about 90% the conversion of l-[3,4-(14)C] methionine to (14)C(2)H(4) suggesting autoinhibition of ethylene production.The results suggest that ethylene biosynthesis in leaves is controlled by hormones, especially auxin, and possibly the rate of ethylene production depends, via a feedback control system, on the rates of ethylene binding at its receptor sites.     

Regeneration in Streptocarpus Leaf Discs and its Regulation by Temperature and Growth Substances

The formation of adventitious buds and roots in leaf discs of Streptocarpus x bybridus‘Constant Nymph’ were both stimulated by relatively low temperatures (12 and 18°C) applied to isolated discs or to the growing plants before leaf harvest. Auxins also promoted both bud and root formation, the optimum concentration for rooting always being one to two orders of magnitude higher than the optimum for budding. Cytokinins had only a small stimulatory effect on bud formation. At higher concentrations it was inhibitory and even counteracted the stimulatory effect of auxin on bud formation. As usual, root formation was inhibited by cytokinin. GA₃ inhibited both bud and root formation but the inhibition was reversible by auxin. In presence of optimum auxin levels abscisic acid enhanced bud formation. It had little effect on root formation except for an inhibition at high concentrations. The effects of exogenous auxin and cytokinin suggest that Streptocarpus leaves have a high and non-limiting level of endogenous cytokinin with auxin as the limiting factor for both root and bud formation. This would also explain the exceptionally high regeneration ability of this plant.     

'早红'草莓高效遗传转化受体系统的建立

本文以草莓主栽品种'早红'组培苗离体叶片和叶柄为外植体,进行叶龄、暗培养、植物生长调节剂配比及抗生素敏感性研究,建立草莓高效遗传转化的受体系统.在含3.0 mg/L 6-BA与0.1 mg/L 2,4-D的MS培养基上,30 d叶龄的叶片再生频率高达98.31%,平均每叶片再生芽数5.09个,叶柄切段的再生频率为89.25%,平均每叶柄切段再生芽数4.92个,叶片的再生频率略高于叶柄;不定芽在含0.2 mg/L 6-BA与0.2 mg/L GA_3的MS继代培养基上培养成苗.将生长状态良好的不定芽转至含0.2 mg/L IBA的1/2 MS培养基上生根,生根率达100%,平均生根数量16.27条,平均根长1.85 cm.抗生素敏感性试验表明,草莓外植体适宜的卡那霉素选择压力为25 mg/L,头孢霉素的筛选浓度为300mg/L.本研究建立的再生体系可作为草莓遗传转化的受体系统.     

Effects of cytokinin and senescence-inducing factors on expression of P ARR5 -GUS gene construct during leaf senescence in transgenic Arabidopsis thaliana plants

ARR5-gene expression was studied in the course of natural leaf senescence and detached leaf senescence in the dark using Arabidopsis thaliana plants transformed with the P _ARR5 -GUS gene construct. GUS-activity was measured as a marker of ARR5-gene expression. Chlorophyll and total protein amounts were also estimated to evaluate leaf senescence. Natural leaf senescence was accompanied by the progressive decline in the GUS-activity in leaves of the 2nd and 3rd nodes studied, and this shift of GUS-activity was more pronounced than the loss of chlorophyll content. The ability of the ARR5-gene promoter to respond to cytokinin was not eliminated during natural leaf senescence, as was demonstrated by a cytokinin-induced increase in GUS activity in leaves after their detachment and incubation on benzyladenine (BA, 5 × 10⁻⁶ M) in the dark. Leaf senescence in the dark was associated with the further decrease in the GUS-activity. The ARR5-gene promoter response to cytokinin was enhanced with the increase of the age of plants, taken as a source of leaves for cytokinin treatments. Hence, although the expression of the ARR5 gene reduces during natural and dark/detached leaf senescence, the ARR5-gene sensitivity to cytokinin was maintained in both cases and even increased with the leaf age. This data suggest that the ARR5 gene, which belongs to the type-A negative regulators of plant response to cytokinin, could be a feedback regulator able to prevent retardation by cytokinin of leaf senescence when it is important for plant life. Growth regulators either reduced ARR5 gene response to cytokinin during senescence of mature detached leaves in the dark (SA, meJA, ABA, SP) or increased it (IAA), thus modifying the resulting rate of its expression.     

Ethylene as a regulator of senescence in tobacco leaf discs

The regulatory role of ethylene in leaf senescence was studied with excised tobacco leaf discs which were allowed to senesce in darkness. Exogenous ethylene, applied during the first 24 hours of senescence, enhanced chlorophyll loss without accelerating the climacteric-like pattern of rise in both ethylene and CO₂, which occurred in the advanced stage of leaf senescence. Rates of both ethylene and CO₂ evolution increased in the ethylene-treated leaf discs, especially during the first 3 days of senescence. The rhizobitoxine analog, aminoethoxy vinyl glycine, markedly inhibited ethylene production and reduced respiration and chlorophyll loss. Pretreatment of leaf discs with Ag⁺ or enrichment of the atmosphere with 5 to 10% CO₂ reduced chlorophyll loss, reduced rate of respiration, and delayed the climacteric-like rise in both ethylene and respiration. Ag⁺ was much more effective than CO₂ in retarding leaf senescence. Despite their senescence-retarding effect, Ag⁺ and CO₂, which are known to block ethylene action, stimulated ethylene production by the leaf discs during the first 3 days of the senescing period; Ag⁺ was more effective than CO₂. The results suggest that although ethylene production decreases prior to the climacteric-like rise during the later stages of senescence, endogenous ethylene plays a considerable role throughout the senescence process, presumably by interacting with other hormones participating in leaf senescence.     

Levels and immunolocalization of endogenous cytokinins in thidiazuron-induced shoot organogenesis in carnation

We evaluated the capacity of the plant growth regulator thidiazuron (TDZ), a substituted phenylurea with high cytokinin-like activity, to promote organogenesis in petals and leaves of several carnation cultivars (Dianthus spp.), combined with 1-naphthaleneacetic acid (NAA). The involvement of the endogenous auxin indole-3-acetic acid (IAA) and purine-type cytokinins was also studied. Shoot differentiation was found to depend on the explant, cultivar and balance of growth regulators. TDZ alone (0.5 and 5.0 micromol/L) as well as synergistically with NAA (0.5 and 5.0 micromol/L) promoted shoot organogenesis in petals, and was more active than N6-benzyladenine. In petals of the White Sim cultivar, TDZ induced cell proliferation in a concentration-dependent manner and, on day 7 of culture, the proportion of meristematic regions in those petals allowed the prediction of shoot regeneration capacity after 30 days of culture. Immunolocalization of CK ribosides, N6-(delta2-isopentenyl)adenosine, zeatin riboside (ZR) and dihydrozeatin riboside (DHZR), in organogenic petals showed them to be highly concentrated in the tips of bud primordia and in the regions with proliferation capacity. All of them may play a role in cell proliferation, and possibly in differentiation, during the organogenic process. After seven days of culture of White Sim petals, NAA may account for the changes found in the levels of IAA and DHZR, whereas TDZ may be responsible for the remarkable increases in N6-(delta2-isopentenyl)adenine (iP) and ZR. ZR is induced by low TDZ concentrations (0.0-0.005 micromol/L), whereas iP, that correlates with massive cell proliferation and the onset of shoot differentiation, is associated with high TDZ levels (0.5 micromol/L). In addition to the changes observed in quantification and in situ localization of endogenous phytohormones during TDZ-induced shoot organogenesis, we propose that TDZ also promotes growth directly, through its own biological activity. To our knowledge, this study is the first to evaluate the effect of TDZ on endogenous phytohormones in an organogenic process.     

Deferral of leaf senescence with calcium

In view of the possibility that senescence may be a consequence of the deterioration of membrane compartments in the cells of leaves, calcium was studied as a possible agent which might defer senescence. The senescence of corn leaf discs was deferred by added calcium, and the effect was additive to the cytokinin deferral of senescence. Likewise, the senescence of Rumex leaf discs was deferred by added calcium, and the effect was additive to the gibberellin deferral of senescence. Detailed experiments with corn leaf discs established that the increase in apparent free space associated with senescence was completely prevented by calcium. An increase in hydraulic permeability during senescence was likewise demonstrated, and this increase was deferred by calcium; calcium plus benzyladenine was even more effective. Each of the measured functions of leaf senescence (chlorophyll content, protein decrease, apparent free space increase, and hydraulic permeability increase) was suppressed by calcium, and the interpretation is offered that the effects are a consequence of the calcium function in maintaining cellular membranes.