An endogenous growth inhibitor, 3-hydroxy-β-ionone. II. Changes in its levels associated with the light-induced growth inhibition of hypocotyls of Phaseolus vulgaris

Dark-grown, 4-day-old seedlings of dwarf bean ( Phuseolus vulgaris L. cv. Morocco) and tall bean (cv. Kentucky Wonder) were transferred to conditions of continuous light at various fluence rates, and the resulting changes in growth rates and concentration of 3-hydroxy-β-ionone, a novel endogenous growth inhibitor, were monitored. The light-induced inhibition of growth and the levels of the inhibitor concentration were dependent on the fluence rate of light applied. The growth rate of hypocotyls of both cultivars decreased rapidly and reached a plateau 18 h after the onset of light. However, the more noticeable changes were the marked inhibition of growth of the hypocotyls of the dwarf cultivar. The concentration of 3-hydroxy-β-ionone in the hypocotyls of both cultivars increased after a 20-min lag and reached plateau values after 12 h. The concentration of the inhibitor in the hypocotyls of the dwarf cultivar increased to about 4 to 8 times the level in the nonirradiated controls, while that in the hypocotyls of the tall cultivar increased only about 2- to 3-fold under the same conditions. The high concentration of 3-hydroxy-β-ionone in the dwarf cultivar under steady-state conditions in continuous light, as compared with that in the tall cultivar, may determine the growth habit of these cultivars.     

An endogenous growth inhibitor, 3-hydroxy-β-ionone. I. Its role in light-induced growth inhibition of hypocotyls of Phaseolus vulgaris

The effects of 3-hydroxy-β-ionone on the growth of hypocotyls of dark-grown dwarl bean ( Phaseolus vulgaris L. cv. Morocco) and tall bean (cv. Kentucky Wonder) were examined. 3-Hydroxy-β-ionone inhibited growth of the hypocotyl of both cultivars at doses higher than 3 ng per plant. Seedlings of the tall cultivar responded to a slightly lesser extent to 3-hydroxy-β-ionone than those of the dwarf cultivar. Changes in the levels of 3-hydroxy-β-ionone in hypocotyls of both cultivars 24 h after the onset of light irradiation, were determined by high-performance liquid chromatography and the results compared with the results of light-induced inhibition of growth. The difference in levels of 3-hydroxy-β-ionone between the two cultivars was considerable, and the changes in its levels in the hypocotyls of each cultivar reflected the light-induced inhibition of growth of the hypocotyls of the respective cultivars. These results suggest that the endogenous growth inhibitor. 3-hydroxy-β-ionone, may play an important role in the inhibition by light of hypocotyl growth of the two cultivars and may serve to distinguish the growth habits of these cultivars.     

表油菜素内酯对绿豆上胚轴内源IAA及其氧化酶的影响

用0.5ppm表油菜素内酯处理绿豆幼苗,显著促进上胚轴伸长生长,若切除真叶则可抑制表油菜素内酯诱导的效应。三碘苯甲酸(TIBA)也可抑制表油菜素内酯促进的伸长生长。外源IAA能部分恢复TIBA的抑制效应。经处理的上胚轴内源IAA含量明显高于对照。暗示表油菜素内酯可能通过对内源IAA的调节来促进绿豆上胚轴的伸长生长。 表油菜素内酯处理的绿豆上胚轴组织中,与生长素降解密切相关的IAA氧化酶以及过氧化物酶活性均明显低于对照。     

Blockage by gibberellic Acid of phytochrome effects on growth, auxin responses, and flavonoid synthesis in etiolated pea internodes

Red light inhibits the growth of etiolated pea internodes, causes a shift toward higher indoleacetic acid (IAA) concentrations in the IAA dose-response curve of excised sections, and promotes the synthesis in intact internodes of kaempferol-3-triglucoside. Gibberellic acid (GA₃) prevents all 3 effects, the first effect substantially and the last 2 completely. This suggests GA₃ blockage of an early or basic event initiated by the active form of phytochrome. The red light-induced shift in the IAA dose-response curve of excised sections is consistent with a light-induced increase in the activity of an IAA destruction system, since the magnitude of the red light inhibition varied with IAA concentration. The red light and GA₃ effects on growth and on flavonoid synthesis are consistent with the view that phytochrome may control growth by regulating the synthesis of phenolic compounds which act as cofactors in an IAA-oxidase system. GA₃ reversal of the red light-induced shift in the IAA dose-response curve involves both growth promotion and inhibition by GA₃ at different IAA concentrations and this, together with the GA₃ reversal of light-induced flavonoid synthesis, supports the suggested regulatory role of phenolic compounds in growth.     

Phytochromabhängige Veränderungen des Wachstums und der Ionenaufnahme etiolierter Erbsenkeimlinge

Summary Inhibition of internodial growth of pea seedlings by light is compensated for by increased growth of leaves. At a given time the sum of fresh weight of internodes plus the product of fresh weight of leaves times a certain factor is constant in darkness or with different periods of light. This correlation may reflect a competition of internodes and leaves for materials delivered at a lightindependent rate from the cotyledons. This hypothesis was tested by immersing roots of pea seedlings into ⁸⁶Rb labelled K-solutions for one day in darkness, removing the plants from the solutions, exposing the seedlings to near or far red light and measuring the radioactivity and fresh weights of leaves and internodes separately. Radioactivity and fresh-weight were both dependent on phytochrome; i.e. inhibition of ion uptake and of growth in internodes and promotion of both processes in leaves by near red light as compared to dark or far red controls are mediated by phytochrome.Short time experiments of ion uptake by the roots show that K transport into the shoot organs is promoted by light after a lag phase of somewhat more than one hour. This interval corresponds well to the lag phase of the light induced growth inhibition of internodes.Seedlings deprived of cotyledons and roots grow well in water but exhibit no difference in growth rate of leaves and internodes in light and darkness. Light dependence is restored if the seedlings are submersed in approximately 3% sucrose solutions. This result seems to indicate that the influence of light on growth rates of leaves and internodes is dependent on the uptake of material by the cell. It seems possible that in the etiolated pea seedling light promotes growth of leaves by promoting uptake and hampers growth of internodes by inhibiting uptake of essential growth material delivered from the cotyledons.     

Effects of compactin,a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor,on the growth of alfalfa (Medicago sativa) seedlings and the rhizogenesis of pepper (Capsicum annuum) explants

The effects of compactin, a specific inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on the growth of alfalfa seedlings in vivo and the rhizogenesis of pepper explants in vitro were investigated. Compactin added to the agar medium inhibited the elongation of roots and hypocotyls of etiolated alfalfa seedlings. The growth inhibition was accompanied by strict inhibition of sterol synthesis. Addition of mevalonic acid, the direct product of 3-hydroxy-3-methylglutaryl coenzyme A reductase, together with compactin relieved the growth inhibition. The sterol level in the seedlings was also protected against the lowering effect of compactin. Similarly, the rhizogenetic process of cultured explants of pepper was inhibited by compactin and relieved by mevalonic acid. Several isoprenoid end products were tested in combination with compactin to determine which compounds, if any, might be limiting for growth. Exogenously supplied isoprenoids failed to relieve the growth inhibition of seedlings. In contrast, they partly relieved the growth inhibition of explants, suggesting their important role in plant growth. During the course of these experiments, it was also found that brassinolide caused remarkable growth inhibition and twisting of alfalfa seedlings.     

Light growth inhibition and growth inhibitors in Sakurajima radish seedlings

Growth of Sakurajima radish seedlings (Raphanus sativus var.hortensis f. gigantissimus Makino) was studied in relation tothe effects of light, the effectiveness of plant hormones, andthe variation in endogenous growth inhibitor content Externally applied GAs and IAA had no effect on the elongationof the hypocotyl in the light as well as in the dark. BA wasslightly promotive in the light, but was not so effective asto nullify the light-induced inhibition of elongation. ABA wasstrongly suppressive in the light as well as in the dark. Growth inhibitors in seedlings were extracted, analyzed by thin-layerchromatography and the Sakurajima radish hypocotyl elongationtest, and 3 acidic and 3 neutral inhibitors were detected. Amongthem all of the acidic and one of the neutral inhibitors increasedwith the time period of illumination, whereas the other 2 neutralsubstances remained almost unchanged in the light but decreasedin the dark. Thus the levels of all 6 inhibitors was higherin light-grown seedlings than in dark-grown ones. This suggeststhat light inhibition in Sakurajima radish hypocotyls may becontrolled by the variation in the inhibitor levels in the seedlings. (Received December 1, 1977; )     

Comparative transcriptional profiling-based identification of raphanusanin-inducible genes

Background  

Raphanusanin (Ra) is a light-induced growth inhibitor involved in the inhibition of hypocotyl growth in response to unilateral blue-light illumination in radish seedlings. Knowledge of the roles of Ra still remains elusive. To understand the roles of Ra and its functional coupling to light signalling, we constructed the Ra-induced gene library using the Suppression Subtractive Hybridisation (SSH) technique and present a comparative investigation of gene regulation in radish seedlings in response to short-term Ra and blue-light exposure.     

Ionones and βIonylideneacetic Acids: Their Influence on Abscisic Acid Biosynthesis by Cercospora rosicola

Several ionones and β-ionylideneacetic acids inhibited absicisic acid (ABA) biosynthesis in Cercospora rosicola at 100 μm. At lower concentrations, α-ionone, 1′,2′-dihydroxy-l′,2′-dihydro-β-ionone and 4′-keto-α-ionone enhanced ABA biosynthesis. Conversions of ionones by C. rosicola were identified by GC-MS as: α-ionone to 4′-keto-α-ionone, 4′-keto-α-ionol and dehydrovomifoliol; and 1′-hydroxy-α-ionone to dehydrovomifoliol. The oxidations of α-ionone and its analogs parallel those of the α-ionylideneacetic acids. The β-ionylideneacetic acids were generally oxidized to more polar forms. Metabolites identified by GC-MS were 3′-hydroxy-, 3′-keto- and 1′,2′-epoxy-1′,2′-dihydro-β-ionylideneacetic acids. The fungus rapidly metabolized most exogenous materials to more polar forms.     

On the role of abscisic Acid and gibberellin in the regulation of growth in rice

Submergence induces rapid elongation of rice coleoptiles (Oryza sativa L.) and of deepwater rice internodes. This adaptive feature helps rice to grow out of the water and to survive flooding. Earlier, we found that the growth response of submerged deepwater rice plants is mediated by ethylene and gibberellin (GA). Ethylene promotes growth, at least in part, by increasing the responsiveness of the internodal tissue to GA. In the present work, we examined the possibility that increased responsiveness to GA was based on a reduction in endogenous abscisic acid (ABA) levels. Submergence and treatment with ethylene led, within 3 hours, to a 75% reduction in the level of ABA in the intercalary meristem and the growing zone of deepwater rice internodes. The level of GA₁ increased fourfold during the same time period. An interaction between GA and ABA could also be shown by application of the hormones. ABA inhibited growth of submerged internodes, and GA counteracted this inhibition. Our results indicate that the growth rate of deepwater rice internodes is determined by the ratio of an endogenous growth promoter (GA) and a growth inhibitor (ABA). We also investigated whether ABA is involved in regulating the growth of rice coleoptiles. Rice seedlings were grown on solutions containing fluridone, an inhibitor of carotenoid and, indirectly, of ABA biosynthesis. Treatment with fluridone reduced the level of ABA in coleoptiles and first leaves by more than 75% and promoted coleoptile growth by more than 60%. Little or no enhancement of growth by fluridone was observed in barley, oat, or wheat. The involvement of ABA in determining the growth rate of rice coleoptiles and deepwater rice internodes may be related to the semiaquatic growth habit of this plant.     

Studies on Abscisic Acid

Epoxidation of methyl dehydro-β-ionylideneacetates with perbenzoic acid afforded methyl 1′, 2′-epoxy-dehydro-β-ionylideneacetates and then methyl 1′, 2′-, 3′, 4′-di-epoxy-dehydro-β-ionylideneacetates. 1′,2′-Epoxy-dehydro-β-ionone, obtained byepoxidation of dehydro-β-ionone, was treated with carbethoxymethylenetriphenlphosphorane to give ethyl 1′, 2′-epoxy-dehydro-β-ionylideneacetates. Further, sensitive photooxidation of ethyl dehydro-β-ionylidenecrotonate, followed by alkaline hydrolysis, gave 1′-hydroxy-4′-keto-α-ionylidenecrotonic acid. Growth inhibitory activities of the above compounds on rice seedlings were examined.     

THE EFFECT OF LIGHT ON THE CELLULAR COMPONENTS OF POLARIZED GROWTH IN BEAN INTERNODES

First internodes of light-grown bean seedlings exposed to supplementary red and far-red light and those of dark-grown seedlings were sectioned and studied to determine the effects of irradiation on the cellular components of polarized growth. Cell counts and measurements of epidermis, cortex, and pith are given. Increased length of internodes of far-red-treated plants was caused by both increased rate and increased duration of cell elongation. The effect of far-red light is interpreted as a reversal of the accelerating effect of light upon cell maturation. It is suggested that investigations of the mechanism of the red, far-red response of stems be concerned with the processes involved in cell elongation. In darkness, rate and duration of cell division as well as rate and duration of cell elongation were greater than in any of the irradiated plants, indicating that only part of the photocontrol of stem elongation is mediated through the red, far-red system.     

Control of 3-hydroxy-3-methylglutaryl coenzyme A reductase by endogenously synthesized sterols in vitro and in vivo.

Isolated rat hepatocytes converted mevalonolactone into sterol intermediates and fatty acids 6- to 8-fold faster than mevalonate salt at concentrations less than 6 X 10(-4) M. Incubation of hepatocytes for 3 h normally results in induction of 3-hydroxy-3-methylglutaryl-CoA reductase. This increase in enzyme activity was inhibited by mevalonolactone and by mevalonate salt; at each concentration between 6 X 10(-4) M and 6 X 10(-8) M the lactone was a more effective inhibitor than the salt. The increase in enzyme activity was completely prevented by 6 X 10(-4) M lactone, and at this concentration the cells synthesized from the lactone an amount of sterol per hour which approximated that leavingthe cells in the same period. Administration of mevalonolactone to intact rats resulted in a dose-dependent inhibition of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase activity. At the highest dose (400 mg of (RS)-mevalonolactone/200 g of rat) enzyme activities declined 85% within 45 min and were still suppressed below normals after 28 h. Mevalonolactone treatment resulted in increases in liver cholesterol content and in the cholesterol ester concentration of liver microsomes. The results demonstrate that the activity of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase can be controlled by the rate of endogenous sterol synthesis both in vitro and in vivo.     

Red-light-induced changes in the distribution of growth inhibitors A-2α and A-2β in dwarf pea seedlings

The distribution of the endogenous growth inhibitors, A-2 and A-2, was determined in light-grown dwarf pea seedlings (Pisum sativum L. cv. Progress No. 9). Red-light-induced changes in this distribution in dark-grown pea seedlings was monitored. The concentration of both inhibitors in light-grown pea seedlings was maximal in the terminal bud and decreased to the lower parts of the seedlings; their concentration in internodes was 7–10 times that in leaves of the same nodes. After the onset of continuous red-light-irradiation, the concentration of the inhibitors in dark-grown pea seedlings increased rapidly in the terminal bud, epicotyl hook and third internode, and the longitudinal gradients of the inhibitors showed a close correlation with the red-light-induced growth inhibition of the stem. The present results suggest that the endogenous growth inhibitors, A-2 and A-2 may be responsible for the red-light-induced growth inhibition of the dwarf pea seedlings.     

Ethylene is not involved in the blue light-induced growth inhibition of red light-grown peas

Although the growth of intact plants is inhibited by irradiation with blue light, the growth rate of isolated stem segments is largely unaffected by blue light. We hypothesized that this loss of responsiveness was a result of ethylene production as part of the wounding response. However, we found no interaction between ethylene- and blue light-induced growth inhibition in dark- or red light-grown seedlings of pea (Pisum sativum L.). Inhibition of growth begins in dark-grown seedlings exposed to blue light within 3 min of the onset of blue light, as was known for red light-grown seedlings. By contrast, ethylene-induced inhibition of growth occurs only after a lag of 20 to 30 min or more (dark-grown seedlings) or 60 min (red light-grown seedlings). Also, the inhibition response of red light-grown seedlings is the same whether ethylene is present from the onset of continuous blue-light treatment or not. Finally the spatial distribution of inhibition following blue light was different from that following ethylene treatment.     

Blue light control of hypocotyl elongation in etiolated seedlings of Lactuca sativa (L.) cv. Grand Rapids related to exogenous growth regulators and endogenous IAA, GA3 and abscisic acid

Results of studies on the interaction of blue light (B) and exogenous applied plant hormones (IAA, GA3 and ABA) as well as inhibitors of their synthesis on the control of hypocotyl elongation in etiolated seedlings of Lactuca sativa (L.) cv. Grand Rapids are presented, and compared with endogenous GA3, IAA and ABA levels measured by capillary gas chromatography-mass spectrometry-selected ion monitoring (GC-MS-SIM). Hypocotyl elongation of etiolated seedlings was linearly inhibited by increasing the B fluence rate between 0 and 18.3 mol m–2 s–1. Both GA3 and IAA added to the incubation medium at different concentrations were able to eliminate partially the inhibition of growth caused by 7.2 mol m–2 s–1 B. When dark (D)-grown seedlings were treated with Ca-prohexadione, a specific inhibitor of gibberellin 3-hydroxylation, they showed a growth inhibition similar to under B. Also, a suppression of growth as in B was obtained when D-grown seedlings were treated with ABA 380 M. By lowering ABA levels with fluridone (an inhibitor of ABA synthesis) a partial reversion of hypocotyl growth inhibition was obtained in B-grown seedlings. While none of the growth promoters used were able to reverse completely the growth inhibition caused by B, a proper combination of GA3, IAA and (eventually) fluridone, abolished the B effects. Correspondingly, lower levels of GA3 and IAA and a higher concentration of ABA were measured by GC-MS-SIM in B-grown hypocotyls than in D-grown ones. These results support the hypothesis that hormones are implicated in mediation of B light-dependent inhibition of hypocotyl elongation, which seems to be the result of a balance among endogenous levels of growth promoting and growth inhibiting hormones.     

Growth and anthocyanin synthesis in excised Sorghum internodes

Summary Ligh-induced anthocyanin synthesis in excised dark-grown internodes of Sorghum was depressed by the addition of auxin to the incubating medium at physiological concentrations. Both IAA and the synthetic auxin, 2,4-D, reduced anthocyanin yield. Similar results were obtained with isolated internode segments and in internodes incubated with coleoptiles (the major source of endogenous auxins) attached. Auxin increased the duration of the lag phase before anthocyanin synthesis began and reduced the rate during the subsequent linear phase. Elongation continued longer with IAA than without it and anthocyanin formation did not begin until extension growth had ceased or was slowing down in both cases; the rate of anthocyanin synthesis in the IAA solution remained depressed compared with that in buffer even after extension growth had ceased in both.At low concentrations IAA stimulated elongation growth without reducing anthocyanin yield and it is unlikely that the effect of IAA on anthocyanin synthesis results from the increased utilisation in growth of substrates needed for anthocyanin formation. The results of reciprocal transfer experiments from dark to light, and vice versa, showed that the action of IAA was associated with its presence in the incubating medium during the irradiation period. If present only in darkness, before or after transfer to light, IAA did not reduce anthocyanin formation; in the former case total yield was increased by IAA as a result of the stimulation of elongation growth, the concentration of anthocyanin remaining unchanged.GA₃ also decreased anthocyanin content; the effect was greater in sections incubated with coleoptiles attached and it is possible that GA₃ acts by increasing the concentration of endogenous auxins. However, CCC, which has been reported to decrease endogenous auxin levels, also reduced anthocyanin yield.The effect of IAA was not influenced by the presence of ascorbate in the incubating medium, nor did added ascorbate result in the formation of any acylated cyanidin derivative in internodes maintained in darkness.Possible relationships between light-induced anthocyanin formation and the photo-inhibition of elongation are discussed.     

Promotion of stem elongation by indole-3-butyric acid in intact plants of Pisum sativum L.

While indole-3-butyric acid (IBA) has been confirmed to be an endogenous form of auxin in peas, and may occur in the shoot tip in a level higher than that of indole-3-acetic acid (IAA), the physiological significance of IBA in plants remains unclear. Recent evidence suggests that endogenous IAA may play an important role in controlling stem elongation in peas. To analyze the potential contribution of IBA to stem growth we determined the effectiveness of exogenous IBA in stimulating stem elongation in intact light-grown pea seedlings. Aqueous IBA, directly applied to the growing internodes via a cotton wick, was found to be nearly as effective as IAA in inducing stem elongation, even though the action of IBA appeared to be slower than that of IAA. Apically applied IBA was able to stimulate elongation of the subtending internodes, indicating that IBA is transported downwards in the stem tissue. The profiles of growth kinetics and distribution suggest that the basipetal transport of IBA in the intact plant stem is slower than that of IAA. Following withdrawal of an application, the residual effect of IBA in growth stimulation was markedly stronger than that of IAA, which may support the notion that IBA conjugates can be a better source of free auxin through hydrolysis than IAA conjugates. It is suggested that IBA may serve as a physiologically active form of auxin in contributing to stem elongation in intact plants.