Endogenous Gibberellin Levels Research Articles

Gibberellic acid‐induced microtubule reorientation in dwarf peas is accompanied by rapid modification of an α‐tubulin isotype

SummaryTo test whether shifts induced in microtubuie orientation by gibberellic acid (GA3) involved changes in tubulin isotypes, pea stem cells were examined when elongation had been enhanced by GA3. The behaviour of a dwarf recessive mutation (le), with very low endogenous levels of gibberellin, was compared with the tall (Le) plant.Two hours after adding GA3, cells were measurably longer than controls and this coincided with a net shift of microtubule orientation from longitudinal and oblique to transverse — an effect that was more pronounced in the dwarf. There were always more cells with net‐transverse microtubules in GA3‐treated tissue than in controls, but as growth ceased, the major orientation of the microtubule arrays became oblique in both samples. Microtubule reorientation was rapid and was closely correlated with the growth of the cells.Although changes in orientation and isotype were monitored over a 40 h period, immunoblotting 2D gels with the well characterized antibodies YL1/2 and YOL1/34 confirmed that alterations to the α‐tubulin constellation could be detected as early as the 2 h time point. Again the effect was especially pronounced in dwarf plants. In the presence of added GA3, one α‐tubulin isotype (designated α1) retained its position in the α‐tubulin constellation (as determined by total protein staining and with YOL1/34 that recognizes detyrosinated as well as tyrosinated tubulin). It was no longer recognized, however, by the anti‐tyrosinated α‐tubulin antibody YL1/2.This indicates that as GA3 begins to cause a reorientation of the cortical microtubules (and to enhance the rate of cell elongation) the α1 isotype is rapidly changed, probably by post‐translational modification.

Effect of paclobutrazol on flower-bud production in Eucalyptusnitens espalier seed orchards

Eucalyptusnitens (Dean & Maiden) Maiden grafts growing as espaliers at Ridgley, Tasmania, and Canberra, Australian Capital Territory, were treated with paclobutrazol as a single collar drench application. Umbel production was enhanced for 3 years at Ridgley and 2 years at Canberra. Canberra grafts were also treated by trunk injection, which proved effective only in the 1st year. Untreated grafts did not produce an appreciable number of umbels until year 3. Paclobutrazol reduced the level of endogenous gibberellins. Close association between gibberellin levels and increases in reproductive activity of E. nitens grafts suggests that the effects of paclobutrazol treatment may be mediated via a reduction in the rate of gibberellin biosynthesis.

Light effects on endogenous levels of gibberellins in photoblastic lettuce seeds

Gibberellin A1 (GA1), 3-epi-GA1 GA17, GA19, GA20, and GA77 were identified by Kovats retention indices and full-scan mass spectra from gas chromatography-mass spectrometry analysis of a purified extract of mature seeds of photoblastic lettuce (Lactuca sativa L. cv. Grand Rapids). Non-13-hydroxylated GAs such as GA4 and GA9 were not detected even by highly sensitive radioimmunoassay. These results show that the major biosynthetic pathway of GAs in lettuce seeds is the early-13-hydroxylation pathway leading to GA1, which is suggested to be physiologically active in lettuce seed germination. Quantification of endogenous GAs in the lettuce seeds by gas chromatography-selected ion monitoring using deuterated GAs as internal standards indicated that the endogenous level of GA1 increased to a level about three times that of dark control 6 h after a brief red light irradiation, and that far-red light given after red light suppressed the effect of red light. The contents of GA20 and GA19 were not affected by the red light irradiation. Evidence is also presented that 3-epi-GA1 is a native GA in the lettuce seeds.

Endogenous Gibberellins in the Developing Liquid Endosperm of Tea

Changes in the kind and level of endogenous gibberellins (GAs) in the developing liquid endosperm of tea (Camellia sinensis L.) were investigated. Gibberellin A1 (GA1), GA8, GA19, GA20, and GA44 were identified by GC-MS or GC-SIM. Besides these early C-13 hydroxylated GAs, GA3, iso-GA3, and GA38 were also identified. Of these GAs, GA1 and GA3 were the major gibberellins. The levels of these GAs were at a maximum in the globular embryo stage and then decreased rapidly during embryo maturation.

Control by Light of Hypocotyl Elongation and Levels of Endogenous Gibberellins in Seedlings of <italic>Lactuca sativa</italic> L.

Control by Light of Hypocotyl Elongation and Levels of Endogenous Gibberellins in Seedlings of <italic>Lactuca sativa</italic> L.

Heat inhibition of tuber development in potato (Solanum tuberosum L.): effects on microtuber formation in vitro

Microtuber formation by single node stem segments of in vitro grown potato plantlets was used as a model system for investigation of heat inhibition of tuber development. The stem segments were incubated in darkness at 20°C or 26°C on a tuberisation medium containing benzyl aminopurine (140 μg 1−1) and sucrose (80 g 1−1). Continuous incubation at 26°C strongly inhibited tuberisation and exposure to 26°C for only 1 week before transfer to 20°C significantly reduced microtuber fresh weight. Inclusion of a gibberellin synthesis inhibitor, paclobutrazol (10−5 M), in the tuberisation medium counteracted the heat inhibition of tuber initiation but not the reduction of microtuber fresh weight. Thus, consistent with previous reports, inhibition of tuber initiation appears attributable to increased endogenous levels of gibberellins. Possible causes of the reduction in microtuber fresh weight are discussed.

The Meristem and Quiescent Centre in Cultured Root Apices of the gib-l Mutant of Tomato (Lycopersicon esculentum Mill.)*

Cultured root apices of tomato bearing the gib-1 mutation, which reduces the levels of endogenous gibberellins, grew slower and were thicker than wild-type controls. This was the result of shorter and broader cells in the meristem of the mutant. Cells of both cortex and stele were affected, but this did not cause any alteration to the volume fraction occupied by these two tissues in the root meristem. Root caps were longer in the mutant and there were also more layers of rhizodermis. All these effects could be reproduced in wild-type roots by addition of 0.1 micromolar 2S,3S paclobutrazol (an inhibitor of gibberellin biosynthesis) to the culture medium and could be normalized in mutant roots by 0.1 micromolar GA3. Cell doubling times in the proximal region of the meristem were similar in mutant and wild-type roots, but were faster in both the quiescent centre (QC) and the cap meristem of the mutant. This latter feature of the mutant roots is likely to be the cause of their longer caps, while the faster rate of division in the QC accounts for the additional tiers of cells that were found to build up in the cortical portion of this zone. These additional tiers failed to form in mutant roots grown in GA3, but they could be induced in wild-type roots by 2S,3S paclobutrazol. These results suggest that endogenous gibberellins may be partly responsible for the slow rate of cell growth and proliferation in the QC.

Stem elongation and changes in the levels of gibberellins in shoot tips induced by differential photoperiodic treatments in the long-day plant Silene armeria

The effects of differential photoperiodic treatments applied to shoot tips and mature leaves of the long-day (LD) plant Silene armeria L. on growth and flowering responses, and on the levels of endogenous gibberellins (GAs), were investigated. Gibberellins were analyzed by gaschromatography-mass spectrometry and the use of internal standards. Exposure of mature leaves to LD, regardless of the photoperiodic conditions of the shoot tips, short days (SD), LD, or darkness, promoted elongation of the stems and of the immature leaves. Long-day treatment of the mature leaves modified the levels of endogenous GAs in shoot tips kept under LD, SD, or darkness. In shoot tips kept in LD or darkness the levels of GA53 were reduced, whereas the levels of GA19 and GA20 were increased. The contents of GA1 were increased in all three types of shoots: SD twofold, LD fivefold, and darkness eightfold. Dark treatment of the shoot tips on plants of which the mature leaves were grown in SD promoted elongation of the immature etiolated leaves and increased the GA1 content of the shoot tips threefold. However, this treatment did not cause stem elongation. The different photoperiodic treatments applied to the shoot tips did not change the levels of GAs in mature leaves. These results indicate that both LD and dark treatments result in an increase in GA1 in shoot tips. In addition, it is proposed that LD treatment induces the formation of a signal that is transmitted from mature leaves to shoot tips where it enhances the effect of GA on stem elongation.

Effects of a Plant-Growth Regulat or, Prohexadione-Calcium (BX-112), on the Endogenous Levels of Gibberellins in Rice

Effects of a Plant-Growth Regulat or, Prohexadione-Calcium (BX-112), on the Endogenous Levels of Gibberellins in Rice

Cellular Growth in Roots of a Gibberellin-Deficient Mutant of Tomato (Lycopersicon esculentumMill.) and its Wild-type

The role of gibberellins in regulating the growth of tomato roots was investigated by comparing various cellular parameters in cultured roots of the gibberellin-deficient mutant gib-l/gib-l with those in roots of the near-isogenic wild-type. In addition, wild-type roots treated with 0·1 μM 2S,3S paclobutrazol, an inhibitor of gibberellin biosynthesis, and mutant roots treated with 0·1 μM GA3 were also compared: the former roots constitute a phenocopy of the mutant, whereas the latter roots appear to be ‘normalized’ and similar to wild-type. The elongation of mutant and phenocopied roots were similar, their maximum elongation rates being about half or two-thirds that of wild-type or GA3-treated mutant roots, respectively. These rates were interpreted in terms of the numbers and lengths of cells within the meristematic and non-meristematic portions of the elongation zone. Mean meristem length tended to be shorter in both the mutant and the 2S,3S paclobutrazol-treated wild-type roots than in the other two types of root. A major difference between the two pairs of mutant and normal roots was their mean final cell lengths: mean lengths of cortical cells of the mutant and 2S,3S paclobutrazol-treated roots were, respectively, 39% and 25% shorter than the mean length of wild-type roots. Final cell length in the GA3-treated mutant roots were similar to wild-type. By contrast, the diameters of mature cortical cells of the mutant and phenocopy were about 20% greater than the diameters of equivalent wild-type or ‘normalized’ mutant cells. The mean volumes of cortical cells in all four types of roots showed no significant differences. Knowledge of the distribution of cortical cell lengths, widths and volumes along the root axis, together with information about the rate of root elongation, permitted comparisons of the relative elemental growth rates of each of these three cellular parameters. The available evidence suggests that the level of endogenous gibberellins in mutant roots is lower than in wild-type roots. The present results, therefore, suggest that endogenous gibberellins are necessary for normal growth of cultured tomato roots and that they regulate the relative amounts of growth at the longitudinal and transverse walls of the cells which, in turn, affects the shape of the elongating cells.

Germination of Tagetes minuta L. II. Role of gibberellins

The effect of the application of gibberellins to Tagetes minuta L. achenes (seeds) was determined at both 25°C, the optimal germination temperature, and 35°C, at which temperature the achenes are thermoinhibited. Both GA3 and GA4+7 accelerated germination at 25°C. Seed germination at 25°C was inhibited by paclobutrazol, but on subsequent application of GA4+7 rapid germination was induced. Following application of GA3 or GA4+7 to thermoinhibited seeds, a significantly higher final germination percentage was observed than in the distilled water control. However, endogenous gibberellin levels in germinating and thermoinhibited seeds did not differ significantly.

Hormonal changes associated with fruit set and development in mandarins differing in their parthenocarpic ability

Satsuma [Citrus unshiu (Mak) Marc.] and Clementine [Citrus reticulata (Hort.) Ex. Tanaka, cv. Oroval] are two related species of seedless mandarins which differ in their tendency to set parthenocarpic fruits. Satsuma fruits naturally set parthenocarpically whereas Clementine mandarins show very low ability to set fruit in the absence of cross‐pollination. The endogenous levels of gibberellins (GAs) and free and conjugated indole‐acetic acid (IAA) and abscisic acid (ABA) throughout early stages of fruit development were investigated in seedless cultivars of both species. Analyses performed by full‐scan combined gas chromatography‐mass spectrometry (GC‐MS) of extracts from ovaries at anthesis demonstrated the presence of GA19, GA20, GA29, GA1, GA8, GA3 and iso‐GA3 in Satsuma mandarin, whereas only GA29, GA3 and trace levels of GA8 were detected in Clementine. At this developmental stage GA‐like substances, as estimated by bioassay, reached their highest levels in Satsuma, while Clementine mandarins contained relatively lower levels. In both species the highest levels of free IAA were found at petal‐fall stage at which time free ABA levels also peaked. Developing fruits of Clementine had higher amounts of both free IAA and ABA. In Satsuma, levels of conjugated IAA remained low throughout reproductive development whereas in Clementine they increased as the free form declined. In contrast, conjugated ABA was at low levels in Clementine but reached higher concentrations in Satsuma. These results suggest that in these mandarins the potential for setting parthenocarpic fruits is mainly influenced by the hormonal status of the fruit during the later stages of cell division and early stages of cell enlargement. Thus, the condition of low ability to set parthenocarpic fruits appears to be associated with lower levels of active GAs, lower capability to catabolize ABA to conjugated ABA and higher ability to conjugate IAA during this period.

Endogenous Levels of Gibberellins, IAA and Cytokinins in <italic>Catharanthus</italic> Crown Gall Tissues of Different Tumor Types

Endogenous levels of gibberellins, auxins and cytokinins in crown gall tissues of Catharanthus roseus G. Don. of two different tumor types, induced by a nopaline-type and an octopine-type Ti-plasmid, were examined

Effect of Temperature on Gibberellin (GA) Responsiveness and on Endogenous GA1 Content of Tall and Dwarf Wheat Genotypes

Near-isogenic wheat (Triticum aestivum L.) lines differing in height-reducing (Rht) alleles were used to investigate the effects of temperature on endogenous gibberellin (GA) levels and seedling growth response to applied GA(3). Sheath and lamina lengths of the first leaf were measured in GA treated and control seedlings, grown at 11, 18, and 25 degrees C, of six Rht genotypes in each of two varietal backgrounds, cv Maris Huntsman and cv April Bearded. Endogenous GA(1) levels in the leaf extension zone of untreated seedlings were determined by gas chromatography-mass spectrometry with a deuterated internal standard in the six Maris Huntsman Rht lines grown at 10 and 25 degrees C. Higher temperature increased leaf length considerably in the tall genotype, less so in the Rht1 and Rht2 genotypes, and had no consistent effect on the Rht1+2, Rht3 and Rht2+3 genotypes. In all genotypes, endogenous GA(1) was higher at 25 degrees C than at 10 degrees C. At 10 degrees C the endogenous GA(1) was at a similar level in all the genotypes (except Rht2+3). At 25 degrees C it increased 1.6-fold in the tall genotype, 3-fold in Rht1 and Rht2, 6-fold in Rht3, and 9-fold in Rht1+2. Likewise, the genotypic differences in leaf length were very conspicuous at 25 degrees C, but were only slight and often unsignificant at 11 degrees C. The response of leaf length to applied GA(3) in the Rht1, Rht2, and Rht1+2 genotypes increased significantly with lowering of temperature. These results suggest the possibility that the temperature effect on leaf elongation is mediated through its effect on the level of endogenous GA(1) and that leaf elongation response to endogenous or applied GAs is restricted by the upper limits set by the different Rht alleles.

A dual-labeling method for identifying differentially expressed genes: use in the identification of cDNA clones that hybridize to RNAs whose abundance in tomato flowers is potentially regulated by gibberellins

A method for identifying cDNA clones that hybridize to differentially expressed RNAs is described. Briefly, the RNA population in which the RNAs of interest are more abundant is used as a template for the synthesis of 35S-labeled cDNAs and another RNA population in which the RNAs of interest are less abundant is used as a template for the synthesis of 32P-labeled cDNAs. The labeled cDNAs are pooled and hybridized to plaque or colony lifts constructed from a cDNA library. Clones that hybridize to RNAs that are differentially expressed are identified using differential autoradiography/fluorography to discriminate between the 32P and 35S isotopes. We have used this method to identify cDNA clones that hybridize to mRNAs that are more abundant in the flowers of wild-type tomato than in the flowers of mutants that have low endogenous levels of gibberellins.

A Gibberellin-Deficient Brassica Mutant—rosette

A single-gene mutant (rosette [ros/ros]) in which shoot growth and development are inhibited was identified from a rapid cycling line of Brassica rapa (syn campestris). Relative to normal plants, the mutant germinated slowly, had delayed or incomplete floral development, and reduced leaf, petiole, and internode growth. The exogenous application of GA(3) by foliar spray or directly to the shoot tip of rosette resulted in rapid flowering, bolting (shoot elongation), and viable seed production. Shoots of rosette contained endogenous levels of total gibberellin (GA)-like substances (;Tan-ginbozu' dwarf rice assay) of about one-tenth of that of the normal rapid-cycling line of B. rapa which consisted almost entirely of a very nonpolar, GA-like substance which yielded GA(1) and GA(3) upon mild acid hydrolysis. In a normal rapid-cycling B. rapa line, the nonpolar putative GA(1) and GA(3) conjugates were present, but additionally, free GA(1) and GA(3) were abundant and identified by gas chromatography-mass spectrometry-selected ion monitoring. The quantities of free GA(1) and GA(3) in the normal line and in rosette were quantified by GC-MS-SIM using [(2)H(2)]GA(1) as an internal standard. Fourteen-day-old rosette and normal seedlings contained 5.3 and 23.2 ng GA(1) per plant, respectively. At day 21 the rosette plants contained 7.7 and 26.1 nanograms per plant of GA(1) and GA(3), while normal plants contained 31.1 and 251.5 nanograms per plant, respectively. Thus, normal plants contained from four to ten times higher levels of total GA-like substances, GA(1), or GA(3), than rosette. The ros allele results in reduced GA level, yielding the rosette phenotype whose delayed germination and flowering, and reduced shoot growth responses indicate a probable role for endogenous GA(1) and GA(3) in the regulation of these processes in Brassica.

The release of the cotyledonary shoot ofpisum sativum from inhibition in relation to changes in the levels of endogenous auxins, cytokinins, and gibberellins

Both axillary buds belonging to the cotyledons (cotyledonary buds) start to grow on decapitated pea seedlings, but one of them (the dominant shoot) prevails in growth over the other (the inhibited shoot). If the dominant' cotyledonary shoot is removed, the inhibited shoot is released from inhibition and starts to grow. This release from inhibition of the inhibited cotyledonary shoot is accompanied within two hours from the removal of the dominant cotyledonary shoot by a marked increase in the level of endogenous cytokinin-like substances and by a decrease in the level of endogenous IAA. By contrast, a significant increase in IAA level and a decreasing trend in the level of cytokinin-like substances occur in the originally inhibited cotyledonary shoot between hour 4 and hour 48 after the release from inhibition of the inhibited cotyledonary shoot. The level of gibberellin-like substances in the cotyledonary shoot released from inhibition steadily increases from the beginning of the release.

EFFECT OF A Rht GENE CONDITIONING THE SEMIDWARF CHARACTER ON WINTERHARDINESS IN WINTER WHEAT (Triticum aestivum L. em Thell)

In the northern part of the North American Great Plains, the level of cultivar winter-hardiness required for winter wheat (Triticum aestivum L.) production is extremely high. Presently, available winter wheat cultivars with adequate winterhardiness are tall and, under favourable growing conditions, crop lodging and excessive amounts of straw can present serious production problems. Consequently, cultivars with short, stiff straw and a high harvest index would be desirable for high production areas within this region. However, semidwarf cultivars with superior winterhardiness have not yet been developed. In this study, six GA-insensitive (Rht) semidwarf parents with poor to moderate winterhardiness were crossed with three GA-sensitive (rht) tall parents possessing good winterhardiness to produce 20 different single, three-way and double crosses. These crosses were evaluated to determine if the GA-insensitive character could be combined with a high level of winterhardiness in winter wheat. Gibberellic acid (GA) sensitivity analyses of F2 seedlings established that a single GA-insensitive gene was involved in each cross. F2-derived F3 and F3-derived F4 lines were assessed for GA-sensitivity and winterhardiness levels were determined from field survival at several locations in Saskatchewan, Canada. Winter survival of homozygous GA-sensitive and GA-insensitive lines were similar in both generations. Lines with winterhardiness levels similar to those of the three tall parent cultivars were recovered in all GA-response classes. The absence of a meaningful pleiotropic effect of Rht genes on winterhardiness indicates that the reason semidwarf cultivars with superior winterhardiness levels have not been developed is due to the lack of a concentrated breeding effort to combine the two characters. A breeding strategy for the production of adapted winterhardy semidwarf winter wheat cultivars is discussed. The influence of endogenous gibberellin levels on cold hardiness in winter wheat is also considered.Key words: Cold hardiness, field survival, Triticum aestivum L, semidwarf, Gibberellic acid

Ethylene as a Mediator of Rubigan Biological Action in Cowpea Plants

AbstractRubigan (a systemic fungicide) inhibited the growth of Rhizoctonia solani in vitro when used at concentrations of 6, 12 and 24 μg/ml respectively. Application of Rubigan as a soil drench significantly reduced the damping‐off incidence on cowpea (Vigna sinensis) plants.Symptoms of growth retardation appearing on cowpea plants treated with Rubigan could be mimiced using an ethylene releasing compound (Ethrel). Ethrel at 10 and 20 μl/ml added to the soil around the seeds induced severe dwarfing as evidenced from measurements of plant length, dry weight and leaf area. Gas chromatographic analysis revealed that ethylene was released either from Rubigan mixed or not with the soil. Rates of ethylene production from soils drenched with Rubigan were relatively inferiorto those produced by authentic Rubigan solutions not added to the soil. Moreover, in all cases ethylene was released at rates proportional to the Rubigan concentrations applied.Factors involved in growth retardation of Rubigan‐treated cowpea plants were discussed in the light of the possible interaction between ethylene and endogenous gibberellin levels.

Fluctuation and localization of endogenous gibberellins in rice.

Changes in the kind and level of endogenous gibberellins (GAs) in the shoot and ear of rice (Oryza sativa L. japonica variety, cultivar Nihonbare) through its life cycle were investigated. Gibberellin A19 (GA19) was the major GA, and GA1, GA20, GA29 and GA53 were minor GAs in the shoot of rice. Besides these 13-hydroxylated (13-OH) GAs, two non-13-hydroxylated (13-H) GAs, namely GA4 and GA34, were identified in the shoot (containing the ear) collected at the heading stage.Further studies on the distribution and changes in the level of GAs in reproductive organs revealed that GA4 was highly concentrated in anthers collected just before anthesis, but was not detected in immature seeds collected about a week after anthesis. These results suggest that 13-H GAs may be biosynthesized in specific organs at a specific stage of the life cycle, and that they may be concerned with the regulation of reproductive growth, while vegetative growth is regulated by 13-OH GAs, probably GA1.