Dose responses of cytokinins

Analysis of published data for the most widely used bioassays for cytokinins reveals that the dose responses are usually subsensitive, i.e. that S₉₀/S₁₀> 81. The data are. in general, insufficiently detailed and/or precise for the kinetics (cooperative, multiphasic or more complex) of the subsensitive responses to be determined. Genus-and species-specific differences in response type were found for callus growth, where the responses are ultrasensitive (S₉₀/S₁₀<81) for tabacco and Phaseolus Iunatus but mostly subsensitive for soybean and P. vulgaris.     

Internode length in Pisum. Gibberellins and the slender phenotype

Pea plants ( Pisum sativum L.) possessing the slender phenotype (conferred by the gene combination la cry^s ) have extremely long, thin internodes and are phenotypically similar to dwarf plants (possessing genes La and/or Cry ) that have been treated with a non-limiting dose of gibberellin (GA₃). In contrast to tall and dwarf plants, slender plants are virtually insensitive to treatment with AMO 1618, PP333 or GA₃ and addition of the "gibberellin-less" mutant gene na does not alter the phenotype of slender plants. Na slender segregates possessed lower levels of gibberellin-like substances than comparable dwarf segregates when extracts from shoots were assayed using the lettuce hypocotyl or rice seedling bioassays. In addition, na slenders possessed little or no gibberellin-like activity even though they possessed a slender phenotype. Thus the gene combination la cry^s causes slender plants to respond as if they are saturated with gibberellins for growth. In addition, the gene combinations la cry^s and le la cry^c (allele cry^c is less extreme in effect than cry^s ) are shown to be almost completely epistatic to the alleles at the na locus. All these results suggest that gibberellin levels are not important in determining the internode length of slender peas (genotype la cry^s ). The possible mechanisms by which this could occur are discussed.     

Correlated responses of chickens to selection for production of antibodies to sheep erythrocytes

A bi directional selection experiment was conducted to measure 5-day antibody titers to sheep erythrocytes in White Leghorn chickens. There was an immediate response to selection with significant differences between lines for the selected trait found in the S₁ and all subsequent generations. Comparisons of S₆, S₇ and S₈ generation females revealed differences between lines in disease resistance and in certain reproductive traits such as age at first egg, percentage hen-day egg production, percentage fertility and duration of fertility. The implications of these correlated responses are important to selection programs for general disease resistance.     

Gibberellin levels and cold-induced floral stalk elongation in tulip

To investigate the role of gibberellins (GAs) in the cold requirement of tulip ( Tulipa gesneriana L. cv. Apeldoorn), bulbs were dry-stored at 5°C or at 17°C for 12 weeks prior to planting at 20°C. Only precooled bulbs showed rapid sprout growth and developed a full-grown flower. Endogenous GA levels were measured in sprouts and basal plates at the time of planting and in the second week after planting, by combined gas chromatography-mass spectrometry using deuterated internal standards. GA₄ was the major gibberellin. while GA₁, GA₉ and GA₃₄ were present in lower amounts. At the time of planting, sprouts from non-cooled bulbs contained significantly more GA₄ and GA₁, per sprout than those from precooled bulbs. Hence, there is no direct correlation between rapid sprout growth after planting and high GA levels at planting. In the second week after planting, floral stalks of precooled bulbs contained 2 to 3 times more GA₄ and its metabolite GA₃₄ per floral stalk and per g fresh weight than those of non-cooled bulbs. The results are discussed with regard to the role of gibberellins in the cold-induced floral stalk elongation of tulip.     

Insulin-stimulated ribosomal protein synthesis in maize embryonic axes during germination

Addition of insulin to maize seed ( Zea mays L. cv. Chalqueño) was found to accelerate germination and seedling growth. Insulin-stimulated maize axes showed enhancement of 35 S-methionine incorporation into ribosomal proteins (rp) and mobilization of S₆ rp mRNA into polysomes. Increase in S₆ rp phosphorylation of the small ribosomal subunit (40S) was observed in 32 P-orthophosphate pulse-labeled experiments when maize axes were stimulated by insulin. Application of either wortmannin or rapamycin, inhibitors of protein kinases of the insulin transduction pathway, abolished the insulin stimulatory effect on S₆ rp phosphorylation and on ribosomal protein synthesis. The above data are interpreted as an indication of the existence of an insulin-stimulated signal transduction pathway in maize tissues that is involved in the regulation of translation.     

Identification and quantification of endogenous gibberellins in apical buds and the cambial region of Eucalyptus

Endogenous gibberellins (GAs) were extracted and purified from apical buds of Eucalyptus nitens (Deane and Maid.) Maid. and the cambial region of E. globulus (Labill.). then analysed by capillary gas chromatography-mass spectrometry. GA₁ GA₁₉ GA₂₀ and GA₂₉ were identified by full scan mass spectra. Kovats retention indices and high resolution selected ion monitoring. Using deuterated internal standards. GA₁. GA₁₉. GA₂₀ and putative GA₂₉ and GA₅₃ were quantified in the apical buds, while GA₄. GA₈. GA₉ and GA₄₄ were shown to be either absent or present at very low levels. From the cambial region. GA₁ and GA₂₀ were quantified at levels of 0.30 ng (g fresh weight)-¹ and 8.8 ng (g fresh weight)-¹ respectively. These data suggest that the early 13-hydroxylation pathway is the dominant pathway for GA biosynthesis in Eucalyptus .     

MICROHETEROGENEITY OF BRAIN CYTOPLASMIC AND SYNAPTOPLASMIC ACTINS

Abstract— Actin present in whole rat brain cytoplasm and in synaptosomes was purified by DNase I affinity chromatography. By use of two-dimensional gels and one-dimensional isoelectric focusing gels, brain actin was shown to be composed of two isomeric forms. By comparison with muscle actins, brain actins were identified as the β and γ isomers. Muscle type α actin is not present in brain. Synaptosomal protein with high affinity for DNase I is primarily composed of β and γ actin, however, two minor synaptosomal proteins, S₁ and S₂, with similar DNase I affinity were also isolated. S₁1 and S₂ have the same apparent molecular weight as whole brain actin, are more acidic than the major actin forms and are distinct from a actin. Relative to β and γ actin, the content of S₁ and S₂ is 3-fOld greater in synaptosomes when compared to similar non-synaptosomal species. The results demonstrate heterogeneity of brain actins and compartmentalization of brain proteins with high affinity for DNase I at the synapse. It was also shown that tubulin has selective affinity for the DNase I-actin complex.     

Internode length in Lathyrus odoratus. Effects of mutants l and lb on gibberellin metabolism and levels

After the application of [¹³C³H]-gibberellin A₂₀ to wild-type (tall) sweet peas ( Lathyrus odoratus L.) labelled gibberellin A₁ (GA₁), GA₈, GA₂₉ and 2-epiGA₂₉ were identified as major metabolities by gas chromatography-mass spectrometry after high performance liquid chromatography. By contrast in genetically comparable dwarf ( II ) plants only labelled GA₂₉ and 2-epiGA₂₉ were produced in significant amounts, although evidence was obtained for trace amounts of labelled GA₁ and GA₈. The apical portions of dwarf plants contained 8–10 times less GA₁ than those of tall plants but at least as much GA₂₀ (measured using di-deuterated internal standards). In conjunction with previous data these results strongly indicate that in genotype ll internode length is reduced and leaf growth altered by a reduction in GA1 levels attributable to a partial block in the 3β-hydroxylation of GA₂₀ to GA₁.
In contrast to dwarf plants, semidwarf plants (genotype lblb ) contained more GA₁ in the apical portion than wild-type counterparts. This is consistent with the suggestion that lb alters some aspect of GA sensitivity.     

The lower limit of photon fluence rate for phototrophic growth: the significance of 'slippage' reactions

Abstract. The role of 'slippage' reactions, in the form of passive H⁺ uniport through CF₀-CF₁, ATP synthetase and breakdown of the S₂ and S₃ intermediates of O₂ evolution, is considered in relation to the growth of phototrophic organisms at low photon fluence rates. Analysis of the limited data available suggests that adaptation (phenotypic or genotypic) to low photon fluence rates is accompanied by an increase in the ratio of light-absorbing pigments to the (potentially slippage-inducing) photosystem two units and CF₀-CF₁ ATP synthetases. Furthermore, organisms which are genotypically adapted to high photon fluence rates do not, when grown at low photon fluence rates, achieve the same low ratio of reaction centres to total light-harvesting pigments as is found in phototrophs genotypically adapted to low photon fluence rates. The limits to, and energy costs of, such a mechanism of adaptation to low photon fluence rates are also discussed.     

Effects of exogenous gibberellins and paclobutrazol on floral stalk growth of tulip sprouts isolated from cooled and non-cooled tulip bulbs

The involvement of gibberellins (GAs) in the regulation of floral stalk elongation and flower development has been studied in tulip. The biological activity of GA₄ and GA₉, both endogenous in tulip bulb sprouts, and GA₁, was tested in vitro on sprouts of cooled and non-cooled tulip bulbs ( Tulipa gesneriana L. cv. Apeldoorn), in the presence or absence of the GA biosynthesis inhibitor paclobutrazol. At early starting dates of incubation, floral stalks from both cooled and non-cooled bulbs hardly showed any elongation in the absence of exogenous GA. Paclobutrazol had no effect on floral stalk elongation, and the response to GAs of sprouts from cooled bulbs was greater than that of sprouts from non-cooled bulbs. At later starts of incubation, considerable floral stalk elongation occurred without GA application. Paclobutrazol inhibited this floral stalk elongation, and the growth of sprouts from both cooled and non-cooled bulbs was stimulated by GA application. The effect of paclobutrazol was reversed by simultaneous application of GA₄ or GA₉. Application of GA with and without paclobutrazol resulted in the same elongation of the floral stalk, indicating the absence of substantial side effects of the inhibitor. The isolated sprouts did not develop a full-grown flower without the addition of GA. GA₄ was more effective than GA₉ in stimulating this flower development. The results demonstrate that both sprouts from cooled and non-cooled bulbs are responsive to exogenous GAs in vitro, and may be a site of GA biosynthesis.     

Interaction of potassium ions and gibberellin in the control of hypocotyl growth in Amaranthus caudatus

Potassium promotes growth in several plant tissues. Elongation growth of the hypocotyls of Amaranthus caudatus L. ev. Lalsag is mainly controlled by gibberellins, but K⁺ also promotes growth. In the present study the interaction of K⁺ with gibberellin (GA₃) and chlorocholine chloride (CCC) has been investigated. When K⁺ was applied externally in the dark, hypocotyl growth was promoted in the seedlings. External application of GA₃ did not promote growth in the dark. GA₃ was effective in the light and K⁺ was synergistic with GA₃ in promoting elongation. Application of CCC in the dark makes the seedlings sensitive to GA₃. The inhibition of growth by CCC was also reversed by K⁺. The results indicate a possible role of K⁺ in GA₃ induced elongation of hypocotyls.     

Embryonal dormancy in apple seeds is controlled by free and conjugated gibberellin levels in the embryonic axis and cotyledons

Halińska, A., Sińska, I. and Lewak, St. 1987. Embryonal dormancy in apple seeds is controlled by free and conjugated gibberellin levels in the embryonic axis and cotyledons.
Free and conjugated gibberellins (GAs) A₄₊₇ and A₉ were determined in embryonic axes and in cotyledons of seeds of apple ( Malus domestica Borb., cv. Antonó wka) during breaking of dormancy under cold stratification. In both organs, the maximum level of free GA₄₊₇ was found at day 30 of stratification, but the concentration was 700 times higher in axes than in cotyledons. Comparison of changes in free and conjugated GA₄₊₇ levels during stratification allow us to suggest that the accumulation of free hormone in axes is, at the most, to 40% due to release from conjugates already present in the axis; that maximally 20% is derived from hydrolysis of cotyledonary conjugates translocated to axes; and that at least 40% originate from the novo biosynthesis of the hormone. Free and conjugated GA₉ levels were similarly altered in axes and in cotyledons, markedly increasing at the end of afterripening. Both release of the free hormone from conjugates and biosynthesis of GA₉, appeared to be involved in that increase; no translocation of free or bound GA₉, between axes and cotyledons was demonstrated.     

An acylcyclohexadione retardant inhibits gibberellin A1 metabolism, thereby nullifying phytochrome-modulation of cowpea epicotyl explants

The regulation by phytochrome of stem elongation in light-grown plants depends on gibberellins (GAs). To investigate whether this is mediated by a change in GA metabolism, the effect of the GA biosynthesis inhibitor LAB 198 999 (an acylcyclohexadione derivative) on the end-of-day far-red (FR) response in cowpea ( Vigna sinensis L.) epicotyl explants has been investigated. Growth of epicotyl explants of light-grown seedlings was enhanced when treated with far-red light before incubation in the dark (end-of-day FR effect). Low doses of LAB 198 999 (0.05 and 0.5 μg explant⁻¹) reduced the effect of FR, whereas 5 to 50 μg explant⁻¹ stimulated elongation of both red light (R)- and FR-treated epicotyl explants while nullifying the differences between R and FR treatments. In paclobutrazol-treated epicotyl explants, FR enhanced the response to applied GA₁ and GA₂₀, whereas LAB 198 999 increased the activity of GA₁ and decreased that of GA₂₀, [³H]Gibberellin A₁, injected into the basal part of the epicotyl, was transported and metabolized mainly to [³H]GA₈ in the apical 20 mm of the epicotyl. The conversion of [³H]GA₁ to [³H]GA₈ was dramatically reduced by both end-of-day FR treatments and LAB 198 999 applications. In addition, both treatments enhanced epicotyl elongation. It is proposed that the regulation of cowpea epicotyl growth by phytocrome is mediated, at least partially, by modifying GA₁ degradation.     

Effects of different gibberellins on elongation growth under short day conditions in seedlings of Salix pentandra

Fifteen different gibberellins (GA's) were tested for their ability to induce elongation growth under short day conditions in seedlings of Salix pentandra L. GA's were applied either to the apex or they were injected into a mature leaf. GA₃ was highly active and also GA₄₊₇ and GA₄ showed high activity. GA₁, GA₂, GA₅, GA₉, GA₁₃, GA₂₀, GA₃₆ and GA₄₇ showed moderate activity. GA₁₆, GA₁₇, GA₂₇ and GA₄₁ exhibited low or no activity in doses up to 10 μg per plant. In general, a better growth response was obtained with an application to the apex than with an injection into the leaf.     

Gibberellin substitution for long day secondary induction of flowering in Poa pratensis

Plants of Poa pratensis cv. Holt initiate inflorescence primordia when exposed to short days (SD) and low temperature, but require a secondary induction by at least 4 long days (LD) for further inflorescence development and stem elongation. Single or double applications of 10 µg per plant of gibberellins A₁, A₃, A₅ and 16,17‐dihydro A₅ (DHGA₅) induced inflorescence development in a high proportion of plants in SD, but only if the plants were detillered to a single stem. Exposure to 2 LD cycles did not cause heading and flowering alone but enhanced the effect of exogenous gibberellins (GAs), bringing flowering to 100%. GA₅ and DHGA₅ were less effective than GA₁ and GA₃ in SD, especially with double applications, but were more effective than GA₁ and GA₃ when given together with 2 LD. The GAs had differential effects on vegetative growth and flowering, GA₅ and DHGA₅ causing much less leaf and stem growth than the other two GAs. Marginal induction, whether by LD or GA application, resulted in a high proportion of spikelets with viviparous proliferation. Thus, whereas GAs are inhibitory to the primary induction by SD, they can replace secondary induction by LD when vegetative growth is limited.     

Immunomodulation of bioactive gibberellin confers gibberellin-deficient phenotypes in plants

Immunomodulation is a means to modulate an organism's function by antibody production to capture either endogenous or exogenous antigens. This method was applied to plants to repress the function of gibberellins (GAs), a class of phytohormones responsible for plant elongation, by anti-bioactive GA antibodies. Two different antibodies were produced in Arabidopsis as single-chain variable fragment (scFv) fused to green fluorescent protein (GFP) with four different subcellular localizations: endoplasmic reticulum (ER), cytosol, apoplastic space or the outer surface of the plasma membrane. When targeting scFv-GFP to ER, plants showed the highest accumulation of scFv-GFP, with binding activity, strong GFP fluorescence in ER-derived compartments and mild but clear GA-deficient phenotypes, including a smaller leaf size, delayed bolting, shorter inflorescence length and decreased germination. Plants expressing scFv-GFP in ER responded to exogenous GA₄ and contained 15–40 times greater endogenous GA₄ than wild-type plants. They also showed increased gene expression for GA3ox1 , GA20ox1 and GA20ox2 , but decreased expression for GA2ox1 , which are feedback and feedforward regulated by GA signalling, respectively. These results suggest that the level of free functional GA₄ decreased when trapped in the ER with scFv to the extent that mild GA-deficient phenotypes were created. A dramatic increase in the total sum of GA₄ (free plus scFv-GFP bound) was detected as a result of the up-regulation of GA biosynthesis (feedback regulated), and a decrease in GA₄ catabolism as a result of protection by scFv-GFP binding. This study demonstrates that the use of immunomodulation to inhibit the action of bioactive GAs is an effective method of creating GA-deficient plants.     

Hormone directed sucrose transport during fruit set induced by gibberellins in Pisum sativum

A new system has been developed to study hormone-directed transport in intact plants during parthenocarpic fruit set induced by gibberellins. Gibberellic acid (GA₃) and gibberellin A₁ (GA₁) applied to unpollinated ovaries of pea ( Pisum sativum L. cv. Alaska) promoted sucrose transport from the leaf to the site of hormone application. In vivo experiments showed an early (30 min) accumulation of [¹⁴C]-sucrose in ovaries of pea stimulated by gibberellins. This activation of sucrose transport appears to be mediated by gibberellins (GA₁, GA₃), increasing both loading of phloem with sucrose in the leaf (source) and sucrose unloading in the ovary (sink). The ability of pea tissue segments to take up sucrose in vitro was not affected by the hormonal treatment.     

BIOASSAY OF ANTHERIDIOGEN IN LYGODIUM JAPONICUM

Antheridia were induced by exogenously applied GA₃ at concentrations between 10⁻⁶ and 3 × 10⁻⁴ M in very young filamentous protonemata of Lygodium japonicum grown in darkness; the longer the dark preculture of protonemata, the lower was the sensitivity of the protonemata to GA₃. Antheridial initials were discernible after 36 hr of GA₃ treatment in the most sensitive protonemata, and the timing of antheridial initiation was delayed with increasing protonemal age.
This quantitative response of the protonemata provided the basis for a new method of assaying gibberellins in terms of the degree of antheridial formation. According to this method, all the gibberellins tested and one of their precursors were active in inducing antheridia in the protonemata, and the activity spectrum of the gibberellins was as follows: GA₇>GA₄>GA₉>GA₃>GA₅>GA₁>GA₈.
The amounts of antheridiogen contained in conditioned media were measured by the present bioassay. A semi-logarithmic relation was shown between the percentage of antheridial formation and the concentration of conditioned medium within a certain dilution range. The amounts of antheridiogen secreted by the prothallia were quantitatively compared by transferring samples onto fresh media for a short period of time.     

Chromatography of acid phytohormones on columns of Sephadex LH-20 and insoluble poly-N-vinylpyrrolidone, and application to the analysis of conifer extracts

The chromatographic behaviour of abscisic acid (ABA), indole-3-acetic acid (IAA), phenylacetic acid (PAA), and gibberellins A₁, A₄, A₈, A₉, A₁₃ and A₂₀ on columns of Sephadex LH-20 and insoluble poly- N -vinylpyrrolidone (PVP) eluted with buffers of different pH values is described. PVP shows considerable batch differences that must be carefully checked. Chromatography of acidic ethyl acetate-soluble fractions of Scots pine ( Pinus sylvestris L.) extracts at pH 4.5 resulted in great losses of phytohormones, due to poor solubility of the extracts. If the extracts were applied to the column dissolved in buffer of pH 7.5, subsequent elution at pH 4.5 was possible with only small losses. The performance of the chromatographic column was strongly affected by the application volume. A combined PVP/Sephadex LH-20 column eluted at pH 4.5 allows remarkable purification of pine and spruce ( Picea abies (L.) Karst.) extracts, collection of IAA in a fraction that can be directly analyzed by e.g. the indolo-α-pyrone method, and collection of another fraction containing ABA, PAA and probably most of the known C₁₉-gibberellins; whereas the C₂₀-gibberellin A₁₃ is eluted later (with IAA).     

Recombinant stigmatic self-incompatibility (S-) protein elicits a Ca2+ transient in pollen of Papaver rhoeas

Evidence for Ca²⁺ signalling in pollen during the self-incompatibility (SI) response in Papaver rhoeas L. has been presented previously. However, it was not known whether the S-protein alone could act as an elicitor of the response or whether the presence of other stigmatic components was required, since relatively crude stigmatic extracts had been used. The S ₁ gene has since been cloned and its product expressed in Escherichia coli has been shown to exhibit biological activity. In this paper it is reported that the recombinant protein (S₁e) elicits a transient rise in [Ca²⁺]_i in incompatible pollen. The Ca²⁺ signal appears indistinguishable from that elicited by S-gene products partially purified from plant extracts in terms of both its timing and spatial distribution. Pollen tube growth is arrested directly after the rise in [Ca²⁺]_i.
The results provide direct evidence that the S-protein alone acts as an elicitor which triggers the Ca²⁺ signal for the pollen SI response. In addition, it is now clear that the recombinant S-protein does not require several post-translational processing events which take place in the plant to act as an elicitor. With respect to the spatial distribution of the Ca²⁺ transient, data are presented which correlate the localized rise in intracellular Ca²⁺ ([Ca²⁺]_i) with the 'nuclear complex' and the endoplasmic reticulum which is associated with this region.