Synergistic interaction of gibberellic acid and triazinone derivatives in the promotion of rice shoot growth

Forty-five 1,3,5-triazine-2,6-dione derivatives were synthesized and their plant growth-promoting activities examined by the rice (Oryza sativa) seedling test in the presence or absence of gibberellic acid (GA). For high activity in promoting the growth of rice seedlings and acting as active GA-synergists, a para-substituted or a 2,4-disubstituted phenyl group, a hydrogen atom and an alkoxy group were required in the 1-, 3- and 4-positions of the 1,3,5-triazine-2,6-dione molecule. 4-Ethoxy-1-(p-tolyl)-s-triazine-2,6(1H, 3H)-dione [TA], one of the most potent triazinones, synergized the effect of GA on the shoot elongation of different varieties of rice including normal type, dwarf mutants and chlorophyll-mutants. TA synergistically increased the growth-promoting activity of GA by both a simultaneous treatment at the same sites and separate treatments at separate sites of rice seedlings.     

Promotion of mesocotyl growth in etiolated rice seedlings by 4-ethoxy-1-(p-tolyl)-s-triazine-2,6(1H,3H)-dione

4-Ethoxy-1-(p-tolyl)-s-triazine-2,6(1H,3H)-dione (TA) promoted mesocotyl growth in dark-grown rice (Oryza sativa L.) seedlings. In cultivars of the japonica type TA alone showed a small promotive effect and TA+gibberellic acid(GA₃) had a marked synergistic effect, while in other cultivars, mostly of the indica type, TA alone showed a great promotive effect and TA+GA₃ had only an additive effect. In cv. Nato, a typical representative of cultivars showing the second type of response, the concentration of TA giving the greatest growth promotion was around 0.1–0.2 mM. In Nato seedlings treated with TA at 0.1 mM, the mesocotyls continued to elongate for 6 days and reached about 75 mm in length, while the mesocotyls of control seedlings grew to a maximum of about 10 mm and growth was limited to the first 3 days after planting. The TA-induced mesocotyl elongation was mainly the consequence of increased cell multiplication in the meristematic area immediately below the coleoptilar node. GA₃, abscisic acid (ABA) and ethylene also stimulated mesocotyl growth in dark-grown Nato seedlings but their effects were much smaller than those of TA. ABA, like GA₃, had an additive effect with TA, but ethylene suppressed the effect of TA and resulted in increased lateral expansion in the upper region of the mesocotyls of TA-treated seedlings.Abbreviations ABA abscisic acid - GA(s) gibberellin(s) - GA₃ gibberellic acid - TA 4-ethoxy-1-(p-tolyl)-s-triazine-2,6(1H,3H)-dione Part 5 in the series Plant Growth-regulating Activities of Isourea Derivatives and Related Compounds; Part 4=Ogawa et al. (1978)     

Isourea and triazinone derivatives as related to their ability to stimulate rice seedling growth

TA [4-ethoxy-1-(p-tolyl)-s-triazine-2,6 (1H, 3H)-dione] wasprepared chemically by intramolecular cyclization of IU [2-ethyl-3-methoxycarbonyl-1-(p-tolylcarbamoyl)-isourea],which has been reported to be an effective GA-synergist. TAalone slightly stimulated the shoot growth of rice seedlings,and in combination with GA showed a distinctly synergistic effecton the growth of rice shoots. The results suggest that TA andIU are nearly equal in their physiological activity. From theresults of the rice seedling test of these two compounds andtheir analogs, structure-activity relationships of isoureaswere correlated with those of triazinones. The results of applicationof IU, at 100 mg/liter to rice seedlings followed by extractionand silica gel thin-layer chromatography, suggested that IUwas biologically converted into its closed-ring form, TA, whichis a stable and potent GA-synergist. Thus, isoureas like IUseem to be easily converted into their counterparts, triazinones,in rice tissues. ¹ This paper is Part III in the series "Plant growth-regulatingactivities of isourea derivatives and related compounds." ² Permanent address: Agricultural Chemicals Research Laboratories,Sankyo Co., Ltd., Hiro-machi, Shinagawa-ku, Tokyo 140, Japan. ³ Present address: Higashi-Osaka Junior College, Nishitsutsumigakuen,Higashi-Osaka 577, Japan. (Received February 21, 1977; )     

Effect of exo-16,17-dihydro-gibberellin A5 on gibberellin A20 metabolism in seedlings of dwarf rice (Oryza sativa L. cv. Tan-ginbozu)

Several of the 16,17-dihydro gibberellins (GAs) inhibit elongation in a variety of species. In a study of their mechanism of action we have investigated the effect of exo-16,17-dihydro-Ga₅ (diHGA₅) on the metabolism of GA₂₀ in dwarf rice (Oryza sativa cv. Tan-ginbozu). A mixture of [³H]- and [³H]-GA₂₀ (100 ng per plant) was applied in microdrops to 4 d old seedlings which were harvested 72 h later. Concurrent treatment with diHGA₅ at 100 ng or 333 ng per plant reduced GA₂₀-induced elongation of the second leaf sheath by 41–66%. There was a concomitant reduction in the amount of [²H₂]GA₁ present at harvest, measured by gas chromatography-mass spectrometry-selected ion monitoring. The [²H₂]GA₂₉ content was also reduced. There was no clear effect of diHGA₅ on the total radioactivity recovered, or on conversion of the [³H]GA₂₀ to putative [³H]GA conjugates, or on the amount of [²H₂]GA₂₀ found. No free [²H₂]GA₈ was detected. In other experiments there was little effect of diHGA₅ on elongation induced by treatment with GA₁. We conclude that diHGA₅ inhibited GA₂₀-induced elongation in dwarf rice shoots at least partly by reducing the 3-hydroxylation of GA₂₀ to GA₁.Abbreviations diHGA₅ = exo- 16, 17-dihydro-gibberellin A₅ - GA = gibberellin - GC-MS-SIM = gas chromatography-mass spectrometry-selected ion monitoring     

Biological activity of some synthetic gibberellin glucosyl esters

Four gibberellin (GA₁, GA₃, GA₄ and GA₃₇) glucosyl esters were synthesized and found to be as active as their respective free acids in the rice seedling bioassay. The rapid hydrolysis of the glucosyl esters in rice seedlings was demonstrated by feeding experiments with glucosyl esters of [³H]GA₁ and [³H]GA₄.     

Synergistic Effect of Isourea and Triazinone Derivatives on Activity of Various Gibberellins

The synergistic effects of 2-ethyl-3-methoxycarbonyl-l-(p-tolylcarbamoyl)-isoureaand 4-ethoxy-l-(p-tolyl)-s-triazine 2,6(1H,3H)-dione on GA_{1,3,4,7,8,9,17,19,20ana 53} in rice seedlings were investigated. Each synergist showeda very high effect when combined with GA_{1,3,9 or 17}, a higheffect with GA_{4,7,19 or 20}, little effect with GA₅₃, and noeffect with GA₈. (Received July 22, 1981; Accepted October 2, 1981)     

The mode of action of a morphactin,fluoren-9-carboxylic acid

Fluoren-9-carboxylic acid acts not only as an auxin but also as an gibberellin-antagonist. In the standard pea straight test (S₅ section) for auxin it stimulated elongation, the optimum concentration being 10 mg/l. On the other hand, it inhibited elongation at 0.1 mg/l. This inhibitory effect was more marked when younger tissue (S₁ section) which also responds to gibberellin was used. Interaction of FCA and IAA in the S₅ section has shown that at higher concentration of IAA there seemed to be a suppraoptimal effect, indicating that FCA acted as an auxin. However, in the S₁ section, the stimulating effect of GA₃ was markedly inhibited by 0.1 mg/l FCA; 10 mg/l FCA was either additive or less than additive to GA₃. In the cucumber hypocotyl test FCA itself was inactive up to 100 μg/plant, but it inhibited the GA₃-induced elongation. This inhibition was overcome by increasing the dosage of GA₃. In the same material, the IAA-induced elongation was not affected by FCA. These results indicate that whether FCA acts as an auxin or a gibberellin-antagonist depends on whether the tissue is sensitive to gibberellin and/or auxin.     

A gibberellin-regulated protein phosphorylated by a putative Ca2+-dependent protein kinase is G-protein mediated in rice root

The GA-signal transduction pathways downstream to the Gα protein in rice seedling root were investigated using in-gel kinase assay and in vitro protein phosphorylation techniques with a Gα protein defective mutant, d1. A 50-kDa protein kinase was detected downstream to Gα protein in the membrane fraction of rice seedling roots using an in-gel kinase assay with histone III-S as a substrate. The activity of a 50-kDa protein kinase increased in the wild-type rice by gibberellin (GA₃) treatment, but did not change in the d1 mutant. This protein kinase activity was inhibited by the Ca²⁺ chelator ethyleneglycol-bis-(beta-aminoethylether)-N,N,N ¹,N ¹-tetraacetic acid (EGTA), protein kinase inhibitors, staurosporine and H7, and calmodulin antagonist, trifluoperazine, suggesting that the 50-kDa protein kinase is a putative plant Ca²⁺-dependent protein kinase (CDPK). The activity of the 50-kDa putative CDPK reached its highest level at 3 h after GA₃ treatment and then gradually declined with time. In order to identify the endogenous substrate for 50-kDa putative CDPK, two-dimensional polyacrylamide gel electrophoresis followed by in vitro protein phosphorylation was carried out. The phosphorylation activity of an endogenous protein PP30, identified as an unknown protein having molecular weight 30 kDa and isoelectric point 5.8 was increased in the wild-type rice by GA₃ treatment, compared with the d1 mutant. The addition of GA₃ treated membrane fraction, which predominantly represent a 50-kDa putative CDPK further increased the phosphorylation of PP30. Almost similar to GA₃ treatment, phosphorylation activity of PP30 was also increased by the treatment with cholera toxin in the wild-type rice but not in d1 mutant. These results suggest that the 50-kDa putative CDPK and an unknown protein, PP30 promoted by GA₃ treatment are G-protein mediated in rice seedling roots.     

Differential effects of camptothecin and interactions with plant hormones on seed germination and seedling growth

Effects of camptothecin, a naturally occurring alkaloid, on seed germination varied from promotive to inhibitory, depending on the species used. It markedly inhibited seedling root growth but its inhibition of hypocotyl growth varied among species. Camptothecin inhibited GA₃-induced dark germination of lettuce (Lactuca sativa L.) seeds and hypocotyl elongation of seedlings. In contrast to ABA, the camptothecin inhibition of GA₃-induced germination could not be overcome by cytokinin. When seeds were germinated at 29C with a 0.5 h light treatment, little or no germination occurred in the camptothecin treatment, but addition of cytokinin overcame this inhibition.     

Identification and characterization of a gibberellin-regulated protein,which is ASR5, in the basal region of rice leaf sheaths

Gibberellins (GAs) regulate growth and development in higher plants. To identify GA-regulated proteins during rice leaf sheath elongation, a proteomic approach was used. Proteins from the basal region of leaf sheath in rice seedling treated with GA₃ were analyzed by fluorescence two-dimensional difference gel electrophoresis. The levels of abscisic acid-stress-ripening-inducible 5 protein (ASR5), elongation factor-1 beta, translationally controlled tumor protein, fructose-bisphosphate aldolase and a novel protein increased; whereas the level of RuBisCO subunit binding-protein decreased by GA₃ treatment. ASR5 out of these six proteins was significantly regulated by GA₃ at the protein level but not at the mRNA level in the basal region of leaf sheaths. Since this protein is regulated not only by abscisic acid but also by GA₃, these results indicate that ASR5 might be involved in plant growth in addition to stress in the basal regions of leaf sheaths.     

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     

Effects of a New Plant Growth Regulator Prohexadione Calcium (BX-112) on Shoot Elongation Caused by Exogenously Applied Gibberellins in Rice (Oryza sativa L.) Seedlings

The effects of a novel plant growth regulator (PGR) prohexadionecalcium (BX-112; calcium 3,5-dioxo-4-propionylcyclohexanecarboxylate)on shoot elongation caused by exogenously applied GA₁, GA₃,GA₄₎ GA₁₉ and GA₂₀ were investigated in rice (Oryza sativa L.cv. Nihonbare and cv. Tan-ginbozu) seedling test. Dependingon the dose, BX-112 reduced shoot elongation in both cultivarscaused by GA₁₉ and GA₂₀, but not by GA₁. When a high dose ofBX-112 (e.g. 250 ng/plant and over) was applied with GA₁, orGA₄, shoot elongation was even promoted. This promotive effect,however, was not observed with GA₃. These results suggest thatBX-112 inhibits gibberellin (GA) biosynthesis in the rice plantat the 3ß- and 2ß-hydroxylation of GAs,namely steps of activation and inactivation, respectively. (Received September 6, 1989; Accepted November 27, 1989)     

Effects of 3-Methylgibberellin Analogs on Gibberellin 3β-Hydroxylases and Plant Growth

Six 3-methylgibberellin analogs were synthesized, and their effects on the GA 3β-hydroxylases from immature seeds of Phaseolus vulgaris and Cucurbita maxima, and/or on the growth of dwarf rice (Oryza sativa L. cv. Tan-ginbozu) and cucumber (Cucumis sativus L. cv. Spacemaster) were investigated. 3-Methyl-GA₅ and 2, 3-didehydro-3-methyl-GA₉· inhibited the conversion of [2, 3-³H2]GA₉ to [2-³H]GA₄ by GA 3β-hydroxylases from both P. vulgaris and C. maxima at 3 μM and higher. Their C/D-ring-rearranged isomers, 2, 3-didehydro-3-methyl-DGC and 16-deoxo-2, 3-didehydro-3-methyl-DGC, inhibited 3β-hydroxylation by the enzyme from P. vulgaris threefold more strongly than the non-C/D-ring-rearranged compounds, but exhibited no effect on 3β-hydroxylation by the enzyme from C. maxima. In a dwarf rice seedling assay, 3-methyl-GA₅ and 2, 3-didehydro-3-methyl-GA₉ promoted shoot elongation at doses of 300 ng/plant and higher, and 3α-methyl-GA₁ and 3α-methyl-GA₄ at doses of 30 ng/plant and higher. In contrast 2, 3-didehydro-3-methyl-DGC inhibited shoot growth to half that of the control at a dose of 300 ng/plant, and 16-deoxo-2, 3-didehydro-3-methyl-DGC showed no effect on growth. In a cucumber seedling assay, 3α-methyl-GA₄ promoted hypocotyl elongation at doses of 300 ng/plant and higher. The other C-3 methyl compounds showed no effect on the hypocotyl elongation of cucumber seedlings.     

Interactions between abscisic acid, ethylene and gibberellin in internodal elongation in floating rice: the promotive effect of abscisic acid at low humidity

The enhancement of internodal elongation in floating or deepwater rice (Oryza sativa L. cv. Habiganj Aman II) by treatment with ethylene or gibberellic acid (GA₃) at high relative humidity (RH) is inhibited by abscisic acid (ABA). Here, we examined the interactive effects of ethylene, gibberellin (GA) and ABA at low RH on internodal elongation of deepwater rice stem segments. Although ethylene alone hardly promoted internodal elongation of stem sections at 30% RH, it enhanced the internodal elongation induced by GA₃. Application of ABA alone to stem segments had no effect on internodal elongation. However, in the presence of ethylene and GA₃ at 30% RH, ABA further promoted internodal elongation. This promotive effect of ABA was not found in the internodes of stem segments treated either with ethylene or with GA₃ at 30% RH or in the internodes of stem segments treated with ethylene and/or GA₃ at 100% RH.     

Light and Electron Microscope Studies on Synergistic Interaction between Gibbereffic Acid and 4-Ethoxy-1-(p-tolyl)-s-triazine-2,6 (1H, 3H)-dione in Growth of Rice Leaf Sheaths

4-Ethoxy-1-(p-tolyl)-s-triazine-2,6(1H,3H)-dione (TA) dramaticallyand synergistically promoted gibberellic acid-induced elongationof rice second leaf sheaths. The elongation from 84 to 132 hafter sowing occurred only at the region 0–2 mm from thebase in control samples, at the 0–4 mm region in TA- orGA-treated samples, and at the 0–12 mm region in TA plusGA-treated samples. The increase in elongation rate inducedby TA and/or GA was greatest in the 0–2 mm region anddecreased gradually toward the ligule. The longitudinal growthinduced by TA and/or GA was due to the increase in cell numbersby cell division, as well as to increase in length of cells.Electron microscopic examinations revealed that TA and/or GAsuppressed the development of plastids which caused the leafcolour to be pale. Irrespective of TA and/or GA treatment(s),microtubules were observed to be exclusively oriented perpendicularto the longitudinal axis of the cell in actively elongatingzones, and in fully elongated zones they were randomly oriented.     

Growth Responses and Allocation of Assimilates of Rice Seedlings by Paclobutrazol and Gibberellin Treatment

Paclobutrazol [(2RS,3RS)-1-(4-chlorophenyl)methyl-4,4-dimethyl-2-(1h-1,2,4-trizol-1-yl)penten-3-ol] effectively decreased vegetative growth of rice (Oryza sativa L.) seedlings and increased the chlorophyll content. The number of veins in a leaf, the calculated number of stomata per leaf, and the length of guard cells were not altered by the paclobutrazol treatment, suggesting an effect on cell elongation. The allocation pattern of carbohydrates was changed by either gibberellin (GA) or paclobutrazol treatment. GA₃ induced more shoot growth and less accumulation of starch than the control and paclobutrazol-treated seedlings. Photosynthetic ability was not affected by either paclobutrazol or GA₃ treatment. Paclobutrazol-treated plants allocated a smaller amount of photosynthates for vegetative shoot growth and stored more as starch in the crowns than the control and GA₃-treated plants. The same starch degrading activity in the crown tissue of paclobutrazol-treated seedlings as in control plants suggests that the accumulated starch is utilized in a normal activity for growth including leaf emergence, tiller formation, and root production, resulting in improved seedling quality. Received May 30, 1996; accepted December 10, 1996     

Growth-active gibberellins overcome the very slow shoot growth of Hancornia speciosa, an important fruit tree from the Brazilian “Cerrado”

Shoot elongation of Hancornia speciosa, an endangered tree from the Brazilian savannah “Cerrado”, is very slow, thus limiting nursery production of plants. Gibberellins (GAs) A₁, A₃, and A₅, and two inhibitors of GA biosynthesis, trinexapac-ethyl and ancymidol were applied to shoots of Hancornia seedlings. GA₁ and GA₃ significantly stimulated shoot elongation, while GA₅ had no significant effect. Trinexapac-ethyl and ancymidol, both at 100 μg per seedling, inhibited shoot elongation up to 45 days after treatment, though the effect was statistically significant only for ancymidol. Somewhat surprisingly, exogenous GA₃ more effectively stimulated shoot elongation in SD-grown plants, than in LD-grown plants. The results from exogenous application of GAs and inhibitors of GA biosynthesis imply that Hancornia shoot growth is controlled by GAs, and that level of endogenous growth-active GAs is likely to be the limiting factor for shoot elongation in Hancornia. Application of GAs thus offer a practical method for nursery production of Hancornia seedlings for outplanting into the field.     

Interaction of growth retardants,daylength, and gibberellins A19, A20, and A1 on shoot elongation in birch and alder

Gibberellins A₁₉, A₂₀, and A₁ were applied to seedlings of birch (Betula pubescens Ehrh.) and alder (Alnus glutinosa (L.) Gaertn.) in order to test their ability to counteract growth inhibition induced by growth retardants (ancymidol and BX-112) or short day (SD, 12 h) photoperiod. Ancymidol inhibits early and BX-112 inhibits late steps in gibberellin biosynthesis. BX-112 inhibited stem elongation in both species while ancymidol, applied as a soil drench, was effective in alder only. Growth retardants affected stem elongation mainly by inhibiting elongation of internodes. All three gibberellins were equally active when applied to seedlings treated with ancymidol; however, only GA₁ was able to counteract the growth inhibition induced by BX-112. SD-induced cessation of elongation growth in birch was counteracted by GA₁, and to some degree, by GA₂₀, while GA₁₉ was inactive. SD treatment did not induce cessation of apical growth in alder. These results are consistent with the hypothesis that of gibberellins belonging to the early C-13 hydroxylation pathway, GA₁ is the only active gibberellin for stem elongation.     

Ethylene-induced stem growth of deepwater rice is correlated with expression of gibberellin- and abscisic acid-biosynthetic genes

Ethylene decreases the content of endogenous abscisic acid (ABA) and increases the level of bioactive gibberellin A₁ (GA₁) in the submerged internodes of deepwater rice. During partial submergence, internodes of deepwater rice undergo rapid elongation as a result of ethylene accumulation in the internodal lacunae. In anin vitro experiment using stem sections from deepwater rice, treatment with 5 μL L^-1 ethylene promoted stem growth by up to 3.2-foId times over air treatment. Expression patterns were analyzed for genes that encode GA- and ABA-biosynthesis enzymes to determine any possible molecular basis for the changes observed in GA₁ and ABA contents as a result of ethylene action. Expression of theOsGA20ox2 andOsGA20ox4 genes, which encode GA 20-oxidase, and of theOsGA3ox2 gene, which encodes the enzyme that converts GA₂₀ to CA₁, was up-regulated, whereas that of three ABA-biosynthetic genes —OsNCED1, OsNCED2, andOsNCEDS-was down-regulated in the presence of ethylene. These results indicate that GA and ABA contribute equally to the submergence-or ethylene-induced stem elongation of deepwater rice via the coordinated and opposite regulation of biosynthesis.