Exogenous GA3 Application Can Compensate the Morphogenetic Effects of the GA-Responsive Dwarfing Gene Rht12 in Bread Wheat

The most common dwarfing genes in wheat, Rht-B1b and Rht-D1b, classified as gibberellin-insensitive (GAI) dwarfing genes due to their reduced response to exogenous GA, have been verified as encoding negative regulators of gibberellin signaling. In contrast, the response of gibberellin-responsive (GAR) dwarfing genes, such as Rht12, to exogenous GA is still unclear and the role of them, if any, in GA biosynthesis or signaling is unknown. The responses of Rht12 to exogenous GA₃ were investigated on seedling vigour, spike phenological development, plant height and other agronomic traits, using F_2∶3 and F_3∶4 lines derived from a cross between Ningchun45 and Karcagi-12 in three experiments. The application of exogenous GA₃ significantly increased coleoptile length and seedling leaf 1 length and area. While there was no significant difference between the dwarf and the tall lines at the seedling stage in the responsiveness to GA₃, plant height was significantly increased, by 41 cm (53%) averaged across the three experiments, in the GA₃-treated Rht12 dwarf lines. Plant height of the tall lines was not affected significantly by GA₃ treatment (<10 cm increased). Plant biomass and seed size of the GA₃-treated dwarf lines was significantly increased compared with untreated dwarf plants while there was no such difference in the tall lines. GA₃-treated Rht12 dwarf plants with the dominant Vrn-B1 developed faster than untreated plants and reached double ridge stage 57 days, 11 days and 50 days earlier and finally flowered earlier by almost 7 days while the GA₃-treated tall lines flowering only 1–2 days earlier than the untreated tall lines. Thus, it is clear that exogenous GA₃ can break the masking effect of Rht12 on Vrn-B1 and also restore other characters of Rht12 to normal. It suggested that Rht12 mutants may be deficient in GA biosynthesis rather than in GA signal transduction like the GA-insensitive dwarfs.     

Growth and gibberellin-A1 metabolism in normal and gibberellin-insensitive (Rht3) wheat (Triticum aestivum L.) seedlings

Growth parameters were determined for tall (rht3) and dwarf (Rht3) seedlings of wheat (Triticum aestivum L.). Plant statures and leaf length were reduced by 50% in dwarfs but root and shoot dry weights were less affected. Leaves of dwarf seedlings had shorter epidermal cells and the numbers of cells per rank in talls and dwarfs matched the observed relationships in overall length. Talls grew at twice the rate of dwarfs (2.3 compared with 1.2 mm h^-1). [³H]Gibberellin A₁ ([³H]GA₁) was fed to seedlings via the third leaf and metabolism was followed over 12 h. Immature leaves of tall seedlings transferred radioactivity rapidly to compounds co-chromatographing with [³H]gibberellin A₈ ([³H]GA₈) and a conjugate of [³H]GA₈, whereas leaves of dwarf seedlings metabolised [³H]GA₁ more slowly. Roots of both genotypes produced [³H]GA₈-like material at similar rates. Isotopic dilution studies indicated a reduced 2-hydroxylation capacity in dwarfs, but parallel estimates of the endogenous GA pool size, obtained by radioimmunoassay, indicated a 12–15 times higher level of GA in the dwarf immature leaves. Dwarfing by the Rht3 gene does not appear to operate through enhanced, or abnormal metabolism of active gibberellins and the act of GA metabolism does not bear an obligate relationship to the growth response.Abbreviations GA_n gibberellin A_n - HPLC high-performance liquid chromatography     

Localization of Stem Elongation Control in Cucumis sativus L

Reciprocal grafts, and applications of gibberellin (GA) and indoleacetic acid (IAA) were used to localize the site of control for stem elongation in cucumber (Cucumis sativus L.). Dwarf and tall plants were reciprocally grafted to determine influence of stems and roots on stem elongation. At 21 days there were no significant differences in length between stems grafted to their own roots and those grafted to roots of the other type. GA₃, GA₄₊₇, and IAA were applied to seedlings with and without live apical buds. Seedlings with live apical buds responded to level of added GA, but not to added IAA. GA₄₊₇ was more effective than GA₃. Hypocotyls of tall plants responded more to both GA treatments than did those of the dwarves when both types had live apical buds. When either GA₄₊₇ or IAA was applied to seedlings with dead apical buds, elongation of the hypocotyl responded to level of the growth regulator, but there was no difference in response between the dwarf and tall plants.     

Inhibition of the growth of peas by tris-(2-diethylaminoethyl)-phosphate trihydrochloride

The effects of Tris-(2-diethylaminoethyl)-phosphate trihydrochloride (SK&F 7997-A₃) on the development of 4 varieties of Pisum sativum were investigated. The compound inhibited shoot elongation of all 4 varieties by as much as 50% or more when seeds were soaked in solutions of the inhibitor for 12 hours before planting. Seed treatment also affected flowering by causing an increase in the number of nodes to the first flower in the early varieties Little Marvel and Alaska. The number of nodes preceding the first flower in the late varieties Dwarf and Tall Telephone was not affected by high concentrations of SK&F 7997-A₃, but low concentrations appeared to cause a slight reduction in the number of nodes to flower.

The inhibitor had little effect on growth when applied to established seedlings; some slight inhibition was noted when high doses were applied to the shoot tip.

SK&F 7997-A₃ suppressed the growth response of dwarf and tall peas to exogenous GA₃. The compound did not inhibit biosynthesis of gibberellin by Fusarium moniliformc when present in shaken liquid cultures at concentrations as high as 10 mg/ml. The inhibitor suppressed the action of applied GA₃ on shoot elongation when the 2 chemicals were applied in 3 ways: 1) inhibitor on lowermost compound leaf and GA₃ on shoot tip; 2) GA₃ on lowermost leaf and inhibitor on shoot tip; and 3) soaking of seeds in the 2 compounds combined for 12 hours prior to planting. The third method of dual treatment yielded evidence that SK&F 7997-A₃ interacts noncompetitively with GA₃ in the regulation of shoot elongation.

     

Contrasting effects of dwarfing alleles and nitrogen availability on mineral concentrations in wheat grain

Background and aim

Concentrations of essential minerals in plant foods may have declined in modern high-yielding cultivars grown with large applications of nitrogen fertilizer (N). We investigated the effect of dwarfing alleles and N rate on mineral concentrations in wheat.

Methods

Gibberellin (GA)-insensitive reduced height (Rht) alleles were compared in near isogenic wheat lines. Two field experiments comprised factorial combinations of wheat variety backgrounds, alleles at the Rht-B1 locus (rht-B1a, Rht-B1b, Rht-B1c), and different N rates. A glasshouse experiment also included Rht-D1b and Rht-B1b+D1b in one background.

Results

In the field, depending on season, Rht-B1b increased crop biomass, dry matter (DM) harvest index, grain yield, and the economically-optimal N rate (N _opt ). Rht-B1b did not increase uptake of Cu, Fe, Mg or Zn so these minerals were diluted in grain. Nitrogen increased DM yield and mineral uptake so grain concentrations were increased (Fe in both seasons; Cu, Mg and Zn in one season). Rht-B1b reduced mineral concentrations at N _opt in the most N responsive season. In the glasshouse experiment, grain yield was reduced, and mineral concentrations increased, with Rht allele addition.

Conclusion

Effects of Rht alleles on Fe, Zn, Cu and Mg concentrations in wheat grain are mostly due to their effects on DM, rather than of GA-insensitivity on N _opt or mineral uptake. Increased N requirement in semi-dwarf varieties partly offsets this dilution effect.     

Sterol composition of dwarf and tall Pisum sativum seedlings in relation to gibberellic-acid-enhanced shoot growth

Gibberellic acid (GA₃) stimulated shoot elongation in both dwarf and tall cultivars of pea, but more so in the dwarf cultivar. The sterol composition of shoots of both cultivars was similar, with sitosterol being the most abundant compound, followed by stigmasterol and campesterol. Cholesterol could not be detected. Following GA₃ application, levels of free sterols in whole shoots increased whereas glycoside levels tended to fall. The magnitudes of the changes in both classes of sterol were similar in both cultivars. Analyses of stems and leaves separately revealed a greater growth response to GA₃ in the former but no effect of the hormone on the sterol composition of either organ. It is concluded that GA₃ enhancement of shoot growth in pea is not mediated through quantitative changes in cell sterols.     

UV-B-induced Inhibition of Stem Elongation and Leaf Expansion in Pea Depends on Modulation of Gibberellin Metabolism and Intact Gibberellin Signalling

Information on the involvement of elongation-controlling hormones, particularly gibberellin (GA), in UV-B modulation of stem elongation and leaf growth, is limited. We aimed to study the effect of UV-B on levels of GA and indole-3-acetic acid (IAA) as well as involvement of GA in UV-B inhibition of stem elongation and leaf expansion in pea. Reduced shoot elongation (13%) and leaf area (37%) in pea in response to a 6-h daily UV-B (0.45 W m^?2) exposure in the middle of the light period for 10 days were associated with decreased levels of the bioactive GA₁ in apical stem tissue (59%) and young leaves (69%). UV-B also reduced the content of IAA in young leaves (35%). The importance of modulation of GA metabolism for inhibition of stem elongation in pea by UV-B was confirmed by the lack of effect of UV-B in the le GA biosynthesis mutant. No UV-B effect on stem elongation in the la cry-s (della) pea mutant demonstrates that intact GA signalling is required. In conclusion, UV-B inhibition of shoot elongation and leaf expansion in pea depends on UV-B modulation of GA metabolism in shoot apices and young leaves and GA signalling through DELLA proteins. UV-B also affects the IAA content in pea leaves.     

Shoot elongation in Lathyrus odoratus L.: Gibberellin levels in light- and dark-grown tall and dwarf seedlings

The levels of gibberellin A₁ (GA₁), GA₂₀, GA₁₉, GA₈, GA₂₉ and GA₈₁ (2-epiGA₂₉) were measured in tall (L-) and dwarf (ll) sweet-pea plants grown in darkness and in light. In both environments the apical portions of dwarf plants contained less GA₁; GA₈ and GA₁₉, but more GA₂₀, GA₂₉, and GA₈₁ than did those of tall plants. It is concluded that the partial block in 3β-hydroxylation of GA₂₀ to GA₁ is imposed by allele l in darkness as well as in the light. Furthermore, darkness does not appear to enhance elongation in sweet pea by increasing GA₁ levels. The reduction of the pool size of GA₁₉ in dwarf plants supports recent theories on the regulation of GA biosynthesis, formulated on the basis of observations in monocotyledonous species. Darkness results in decreased GA₂₀, GA₂₉, and GA₈₁ levels in the apical portions of tall and dwarf plants and possible reasons for this are discussed.     

Gibberellins and leaf expansion in near-isogenic wheat lines containing Rht1 and Rht3 dwarfing alleles

In near-isogenic lines of winter wheat (Triticum aestivum L. cv. Maris Huntsman) grown at 20° C under long days the reduced-height genes, Rht1 (semi-dwarf) and Rht3 (dwarf) reduced the rate of extension of leaf 2 by 12% and 52%, respectively, compared with corresponding rht (tall) lines. Lowering the growing temperature from 20° to 10° C reduced the rate of linear extension of leaf 2 by 2.5-fold (60% reduction) in the rht3 line but by only 1.6-fold (36% reduction) in the Rht3 line. For both genotypes, the duration of leaf expansion was greater at the lower temperature so that final leaf length was reduced by only 35% in the rht3 line and was similar in the Rht3 line at both temperatures. Seedlings of the rht3 (tall) line growing at 20° C responded positively to root-applied gibberellin A₁ (GA₁) in the range 1–10 μM GA₁; there was a linear increase in sheath length of leaf 1 whereas the Rht3 (dwarf) line remained unresponsive. Gibberellins A_{1, 3, 4, 8, 19, 20, 29, 34, 44} and ₅₃ were identified by full-scan gas chromatography-mass spectrometry in aseptically grown 4-d-old shoots of the Rht3 line. In 12-d-old seedlings grown at 20° C, there were fourfold and 24-fold increases in the concentration of GA₁ in the leaf expansion zone of Rht1 and Rht3 lines, respectively, compared with corresponding rht lines. Although GA₃ was present at a similar level to GA₁ in the rht3 (tall) line it accumulated only fivefold in the Rht3 (dwarf) line. The steady-state pool sizes of endogenous GAs were GA₁₉ ? GA₂₀ = GA₁ in the GA-responsive rht3 line whereas in the GA non-responsive Rht3 line the content of GA₁₉≈ GA₂₀ ? GA₁. It is proposed that one of the consequences of GA₁ action is suppression of GA₁₉-oxidase activity such that the conversion of GA₁₉ to GA₂₀ becomes a rate-limiting step on the pathway to GA₁ in GA-responsive lines. In the GA-non-responsive Rht lines it is suggested that GA₁₉ oxidase is not downregulated to the same extent and GA₁ accumulates before the next rate-limiting step on the pathway, its 2β-hydroxylation to GA₈. The steady-state pool sizes of GA_{19, 20, 1, 3} and ₈ were similar in developmentally equivalent tissues of the rht3 (tall) line growing at 10° C and 20° C despite a 2.5-fold difference in the rate of leaf expansion. In contrast, in the Rht3 (dwarf) line, the extent of accumulation of GA₁ reflected the severity of the phenotype at the two temperatures with slower growing tissues accumulating less, not more, GA₁. These results are interpreted as supporting the proposed model of regulation of the GA-biosynthetic pathway rather than previous suggestions that GA₁ accumulates in GA-insensitive dwarfs as a consequence of reduced growth rates.     

Genotypic variation in the responsiveness to GA3 within tall (rht1) and semi-dwarf (Rht1) spring wheat

The effect of GA₃ on coleoptile-and first leaf elongation of tall (rht1) and semi-dwarf (Rht1) nearly-isogenic genotypes, within each of 25 random F₉ wheat families, was determined on seedlings grown in a growth room at 18 °C. Conspicuous and very significant inter-family variation in the response of the first leaf to GA₃ application was found in both the rht1 and Rht1 genotypes. The magnitudes of the response of the different families within genotypes to GA₃ were not related to the leaf length of their untreated seedlings. It is suggested that, under given environmental conditions, background genotypic effects, inducing inter-family variation in responsiveness to GA₃, regulate the elongation growth up to the limits set by the Rht alleles.     

Identification and characterization of gibberellin-insensitive mutants selected from among dwarf mutants of rice

In rice, many dwarf mutants have been isolated and characterized. We have investigated the relationship between dwarfism and the gibberellin (GA)-mediated control of physiological processes. Twenty-three rice cultivars and mutants (9 normal, 3 semi-dwarf, 11 dwarf) were analyzed in terms of two GA-mediated processes, namely, elongation of shoots and production of -amylase activity in the endosperm. As a result, we identified four different groups (groups N, T, D and E). Two-dimensional plotting of the extent of induction of -amylase in the endosperm versus the extent of enhancement of shoot elongation upon treatment with exogenous gibberellic acid (GA₃) provided a useful method for the rapid allocation of large numbers of dwarf mutants of rice to the various groups. Members of group N (normal type), which included all normal cultivars and semi-dwarf mutants, showed a slight increase in elongation of shoots and a remarkable increase in production of -amylase with the application of GA₃ during germination. All of the dwarf mutants were classified as being members of the other three groups. Members of group T (Tan-ginbozu type), including three dwarf mutants, were highly responsive to exogenous GA₃ in terms of elongation of shoots and production of -amylase, with associated lower levels of endogenous GA. In contrast, members of the other three groups, including group N, had normal levels of endogenous GAs. Members of group D (Daikoku type) were only slightly responsive to exogenous GA₃, an indication that they are GA-insensitive mutants. Members of group E (Ebisu type) had responses to GA₃ similar to those of group N, not only in terms of elongation of shoots but also in terms of -amylase production, an indication that they are dwarf mutants that can be considered as neither GA-deficient nor GA-insensitive mutants. We also examined a GA-insensitive mutant selected from among 19 near-isogenic dwarf lines of Shiokari, and we concluded that the d-1 gene is associated with the phenotype of GA-insensitive dwarf mutants.     

Growth retardant activity of paclobutrazol enantiomers in wheat seedlings

The resolved enantiomers of paclobutrazol appeared to have different primary modes of action as plant growth retardants in rht3 (tall) wheat seedlings. 2S,3S-Paclobutrazol reduced shoot growth more effectively than root growth, whereas the opposite was the case with the 2R,3R-enantiomer. Low concentrations (0.03–1.0 M) of 2S,3S-paclobutrazol specifically inhibited gibberellin A₁ (GA₁) production in Rht3 (dwarf) seedlings without affecting shoot growth, confirming that inhibition of GA biosynthesis is the primary mode of action of this enantiomer. Reductions in shoot growth of rht3 (tall) wheat treated with 2S,3S-paclobutrazol were associated with reductions in GA₁ content, an effect that could be reversed by gibberellic acid (GA₃) application, showing that GAs are important regulators of light-grown shoot growth in wheat. The inhibition of root growth of wheat seedlings following treatment with 2R,3R-paclobutrazol was associated with a decline in de novo synthesis of major sterols, a decrease in stigmasterol: sitosterol ratio and an accumulation of the 14-methyl sterol, obtusifoliol. Concentrations >3 M 2S,3S-paclobutrazol also affected de novo sterol production in wheat roots, suggesting that root growth is more responsive to interference with sterol than GA biosynthesis. There was a decline in abscisic acid (ABA) content in Rht3 (dwarf) shoots treated with relatively high concentrations of 2S,3S-paclobutrazol but no effect with its optical isomer.     

Promotion of leaf sheath growth by gibberellic acid in a dwarf mutant of rice

The mechanism of gibberellin (GA)-induced leaf sheath growth was examined using a dwarf mutant of rice (Oryza sativa L. cv. Tan-ginbozu) treated in advance with an inhibitor of GA biosynthesis. Gibberellic acid (GA₃) enhanced the growth of the second leaf sheath, but auxins did not. Measurement of the mitotic index and cell size revealed that cell elongation rather than cell division is promoted by GA₃. Gibberellic acid increased the extensibility of cell walls in the elongation zone of the leaf sheath. It also increased the total amount of osmotic solutes including sugars in the leaf sheath, but did not increase the osmotic concentration of the cell sap, due to an accompanying increase in cell volume by water absorption. In the later stage of GA₃-induced growth, starch granules completely disappeared from leaf sheath cells, whereas dense granules remained in control plants. These findings indicate that GA enhances cell elongation by increasing wall extensibility, osmotic concentration being kept unchanged by starch degradation. Received: 28 August 1997 / Accepted: 16 October 1997     

The Role of Gibberellins in Early Growth and Development of Sugar Beet

Two gibberellin(GA)-like compounds were found in both rootsand shoots of sugar beet plants using the barley endosperm bioassay.One had chromatographic properties similar to GA₃ and GA₁; theother was highly non-polar, relatively inactive in the endospermassay, and may be a new gibberellin. Presence of the GA_3/1-likecompound was confirmed with the dwarf rice bio-assay. The quantityof this GA was relatively high in the root compared with theshoot at the 3–4 leaf stage when the first supernumerarycambia are being formed in the root. As plants developed throughthe 8–9 leaf stage and the 15–16 leaf stage thequantity of GA per unit fresh weight of material decreased. Application of gibberellic acid (GA₃) to the roots of youngsugar beet plants caused a significant increase in root dryweight shortly after treatment and the rate at which supernumerarycambia were produced was increased. Application of GA₃ to asingle petiole caused a significant increase in both root andshoot dry weight. GA₃ applied to either root or shoot causeda reduction in the rate of leaf formation although total leafarea per plant and shoot dry weight were unaffected. The probablerole of GA-like substances in controlling the growth and developmentof young sugar beet plants is discussed.     

Gibberellins and Heterosis in Maize : II. Response to Gibberellic Acid and Metabolism of [H]Gibberellin A(20)

Two maize inbreds, CM7 and CM49, and CM7 × CM49, their F₁ hybrid (which displayed significant heterosis), were examined with regard to response to exogenous gibberellin A₃ (GA₃), and in their ability to metabolize GA₂₀, a native GA of maize. The leaf sheath elongation response to GA₃ was far greater for the imbreds than for their hybrid. The inbreds also displayed significant elongation of the leaf blades in response to GA₃, whereas the hybrid was unaffected. Promotion of cell division in the leaf sheath of CM7 and the hybrid was effected by GA₃, but no promotion of cell elongation was observed in CM49, even though significant leaf sheath elongation occurred. Shoot dry weight of both inbreds was significantly increased by GA₃, but response by the hybrid in this parameter was slight and variable. Root dry weight of CM7 was significantly increased by GA₃, but was unchanged in CM49 and the hybrid. Thus, inbred shoot dry weight increases effected by GA₃ were not at the expense of the root system. Rapid metabolism of [2,3-³H]GA₂₀ occurred in all genotypes, although genotypic differences were observed. The hybrid had the highest rates of metabolism to GA glucosyl conjugate-like substances. Oxidative metabolism was also fastest in the hybrid, followed by CM7, and slowest in CM49, the slowest-growing inbred. Thus, rate of GA₂₀ metabolism is under genetic control in normal (i.e. not dwarfed) maize genotypes. These results, taken together with previous reports that the hybrid has significantly enhanced levels of endogenous GA-like substances, suggest that GA play a role in the expression of heterosis in maize.     

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 GA₃ 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 GA₁ and GA₃ upon mild acid hydrolysis. In a normal rapid-cycling B. rapa line, the nonpolar putative GA₁ and GA₃ conjugates were present, but additionally, free GA₁ and GA₃ were abundant and identified by gas chromatography-mass spectrometry-selected ion monitoring. The quantities of free GA₁ and GA₃ in the normal line and in rosette were quantified by GC-MS-SIM using [²H₂]GA₁ as an internal standard. Fourteen-day-old rosette and normal seedlings contained 5.3 and 23.2 ng GA₁ per plant, respectively. At day 21 the rosette plants contained 7.7 and 26.1 nanograms per plant of GA₁ and GA₃, 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, GA₁, or GA₃, 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 GA₁ and GA₃ in the regulation of these processes in Brassica.     

Dwarf mutants ofBrassica: Responses to applied gibberellins and gibberellin content

Eight rapid-cyclingBrassica genotypes differing in height were treated with gibberellins (GAs) by syringe application to the shoot tip. The height of two genotypes ofBrassica napus, Bn5-2 and Bn5-8, andB. rapa mutants,dwarf 1 (dwf1) anddwarf 2 (dwf2), was unaffected by exogenous GA₃ at dosages up to 0.1 μg/plant, a level which increased shoot elongation of normal genotypes. Thus, these dwarf mutants are “GA-insensitive.” In contrast to theB. napus dwarfs, twoB. rapa mutants,rosette (ros), anddormant (dor), elongated following GA₃ application. The dwarfros was most sensitive, responding to applications as low as 1 ng GA₃/plant. Furthermore,ros also responded to GA₁ and some of its precursors with decreasing efficacy: GA₃>ent-kaurenoic acid ≥GA₁>GA₂₀≥GA₁₉=GA₄₄≥GA₅₃. Endogenous GAs were measured by gas chromatography-selected ion monitoring using [²H₂]GA internal standards for calibration, from shoots of the GA-insensitive genotypes Bn5-2, Bn5-8 which contained theB. napus mutantdwarf 1, and from a normal genotype Bn5-1. Concentrations of GA₁ and GA₂₀ averaged 3.2- and 4.6-fold higher, respectively, and GA₁₉ levels also tended to be higher in the dwarfs than in the normal genotype.