Effect of thidiazuron, a cytokinin-active urea derivative, in cytokinin-dependent ethylene production systems

Cytokinins are known to stimulate ethylene production in mungbean hypocotyls synergistically with indoleacetic acid (IAA), in mungbean hypocotyls synergistically with Ca²⁺, and in wilted wheat leaves. Thidiazuron, a substituted urea compound, mimicked the effect of benzyladenine (BA) in all three systems. In the Ca²⁺ + cytokinin system and the IAA + cytokinin systems of mungbean hypocotyls, thiadiazuron was slightly more active than BA at equimolar concentration. In mungbean hypocotyls exogenously applied IAA was rapidly conjugated into IAA asparate, and this conjugation process was effectively inhibited by thidiazuron, as by cytokinins. In the wilted wheat leaves system, 10 micromolar thidiazuron exerted stress ethylene production equal to that exerted by 1 millimolar BA, indicating that thidiazuron is more active than BA by two orders. The structure-activity relationship of thidiazuron and its thiadiazolylurea analogs in stimulating Ca²⁺-dependent ethylene production in mungbean hypocotyls was found to agree well with the structure-activity relationship of these derivatives in promoting the growth of callus tissues. These results indicate that thidiazuron and its derivatives are highly active to mimic the adenine-type cytokinin responses in promoting ethylene production and that the structure-activity relationship in promoting the growth of callus and in promoting ethylene production is similar.     

Light promotes the synthesis of lignin through the production of H2O2 mediated by diamine oxidases in soybean hypocotyls

In order to analyze the relationship between polyamine oxidative degradation induced by light and the lignin synthesis in cell walls, the activities of diamine oxidases and peroxidase, the contents of H₂O₂ and lignin, and the growth of hypocotyls in soybean [Glycine max (Linn.) Merr.] grown under light or in darkness were investigated. In comparison with the dark treatment, light irradiation significantly inhibited the growth of soybean hypocotyls and promoted the activities of diamine oxidases and peroxidase as well as the accumulation of H₂O₂ and lignin. Treatments with the different concentrations of diamine oxidase inhibitors (2-hydroxyethylhydrazine and aminoguanidine) under the light condition inhibited diamine oxidase activity, and decreased the contents of H₂O₂ and lignin. The results provide evidence for the hypothesis that light irradiation could promote the accumulation of H₂O₂ and lignin in cell walls by activating polyamine oxidative degradation mediated by diamine oxidases.     

The interaction of light irradiance with auxin in regulating growth of <Emphasis Type="Italic">Helianthus annuus</Emphasis> shoots

Plants growing under canopy shade or in near-neighboring proximity of taller vegetation are the receivers of shade light conditions. The effect of light irradiance (photosynthetically active radiation [PAR]), one of the main components of shade light, on the growth of various tissues of sunflower seedlings and the possible role of auxin were investigated. Gradual reductions in PAR irradiance level from near-normal to low and very low result in significant and gradual increases in sunflower hypocotyl growth and endogenous auxin content. Similar reductions in PAR level resulted in significant and gradual decreases in sunflower cotyledon and leaf growth, and endogenous auxin content. Exogenously applied auxin increased hypocotyl elongation under near-normal PAR, where IAA levels are below optimum, but decreased elongation under very low PAR, where IAA levels are already at optimum. These results suggests that auxin acts as positive growth regulator of sunflower hypocotyls subjected to low light irradiance stress. This is further supported by the transfer experiments where seedlings transferred, for example, from near-normal PAR to very low PAR showed increased elongation associated with increased IAA levels. Therefore, it is reasonable to conclude that light irradiance-mediated changes in hypocotyl elongation of young sunflower seedlings are regulated by endogenous auxin levels.     

The Effect of Epibrassinolide on Arabidopsis Seedling Morphogenesis and Hormonal Balance under Green Light

We studied the effect of 24-epibrassinolide (EB) on the levels of endogenous hormones and photomorphogenesis of Arabidopsis thaliana (L.) Heynh wild-type (Ler) and mutant (hy4) seedlings. This mutant is deficient in the cryptochrome 1 (CRY1) synthesis. CRY1, which is a product of the HY4 gene, is a blue light photoreceptor in wild-type plants, but is sensitive to green light as well. In dark-grown seven-day-old mutant seedlings, the ABA/zeatin ratio differed from this ratio in wild-type seedlings. Thehy4 mutant exhibited a lower zeatin and higher free-ABA contents, which could retard its hypocotyl growth in darkness. EB retarded the growth of hypocotyls in etiolated hy4 seedlings and enlarged their cotyledons more efficiently than in wild-type seedlings. Green light (GL) did not affect the growth of hypocotyls but enlarged cotyledons of hy4 seedlings, which might be associated with some increase in the level of free IAA and a considerable decrease in free ABA and also with a decrease in the cytokinin level in seedlings. The hy4 cotyledon response to GL depended evidently on photoreceptors other than CRY1. GL enhanced the effects of EB on the morphogenesis of both Ler and hy4 seedlings, which was coupled with changes in the balance of endogenous IAA, ABA, and cytokinins. We may suppose that EB is involved in the control of photomorphogenesis by interaction with endogenous hormones, which are involved in the transduction of a light signal absorbed by the GL photoreceptors.     

Role of extensin peroxidase in tomato (Lycopersicon esculentum Mill.) seedling growth

 It is proposed that inhibition of extensin peroxidase activity leads to a less rigid cell wall and thus promotes cell expansion and plant growth. A low-molecular-weight inhibitor derived from the cell walls of suspension-cultured tomato cells was found to completely inhibit extensin peroxidase-mediated extensin cross-linking in vitro at a concentration of 260 μg/ml. The inhibitor had no effect upon guaiacol oxidation catalyzed by extensin peroxidase or horseradish peroxidase. We have demonstrated that the light-irradiated inhibition of plant growth may be partially offset by inhibition of endogenous extensin peroxidase activity. Overall plant growth was enhanced by up to 15% in the presence of inhibitor relative to control plants. Inhibitor-treated and illuminated tomato hypocotyls grew up to 15% taller than untreated controls. The inhibitor had no effect upon etiolated plants over a 15-d period, suggesting that only low levels of peroxidase-mediated cross-linking can be found in the cell walls of etiolated plants. SDS-PAGE/Western blots of ionically bound protein from both etiolated and illuminated hypocotyls identified a doublet at 57/58.5 kDa which is immuno-reactive with antibodies raised to tomato extensin peroxidase. Levels of the 58.5-kDa protein, determined by SDS-PAGE, were at least threefold higher in illuminated tomato hypocotyls than in etiolated hypocotyls. Three fold higher levels of extensin peroxidase, elevated in-vitro extensin cross-linking activity and 15% higher levels of cross-linked, non-extractable extensin were observed in illuminated tomato hypocotyls compared with etiolated tomato hypocotyls. This suggests that white-light inhibition of tomato hypocotyl growth appears to be mediated, at least partially, by deposition of cell wall extensin, a process regulated by M_r-58,500 extensin peroxidase. Our results indicate that the contribution of peroxidase-mediated extensin deposition to plant cell wall architecture may have an important role in plant growth. Received: 22 July 1999 / Accepted: 11 October 1999     

Endogenous Phytohormones and Regulation of Morphogenesis of Arabidopsis thalianaby Blue Light

We studied the effects of blue light (BL) on the levels of endogenous phytohormones (IAA, ABA, gibberellins, and cytokinins) and morphogenesis of the 7-day-old Arabidopsis thaliana(L.) Heynh seedlings of wild type (Ler) and its hy4mutant with a disturbed synthesis of cryptochrome 1 (CRY1), which is a receptor for BL. In darkness, the mutant contained considerably less free IAA and zeatin, but much more ABA as compared to the wild-type seedlings. BL retarded the hypocotyl growth in the wild-type seedlings but stimulated it in the mutant. Elongation of mutant hypocotyls was accompanied by accumulation of free IAA and a decrease in the content of free ABA; the level of cytokinins did not change. We believe that the response of the hy4hypocotyls to BL is mediated by a BL receptor distinct from cryptochrome 1. The conclusion is that light and hormonal signals interact in the control of the hypocotyl growth in A. thalianaseedlings.     

The Effects of Exogenous Auxins on Endogenous Indole-3-Acetic Acid Metabolism (The Implications for Carrot Somatic Embryogenesis)

The effect of auxin application on auxin metabolism was investigated in excised hypocotyl cultures of carrot (Daucus carota). Concentrations of both free and conjugated indole-3-acetic acid (IAA), [2H4]IAA, 2,4-dichlorophenoxyacetic acid, and naphthaleneacetic acid (NAA) were measured by mass spectroscopy using stable-isotope-labeled internal standards. [13C1]NAA was synthesized for this purpose, thus extending the range of auxins that can be assayed by stable-isotope techniques. 2,4-Dichlorophenoxyacetic acid promoted callus proliferation of the excised hypocotyls, accumulated as the free form in large quantities, and had minor effects on endogenous IAA concentrations. NAA promoted callus proliferation and the resulting callus became organogenic, producing both roots and shoots. NAA was found mostly in the conjugated form and had minor effects on endogenous IAA concentrations. [2H4]IAA had no visible effect on the growth pattern of cultured hypocotyls, possibly because it was rapidly metabolized to form inactive conjugates or possibly because it mediated a decrease in endogenous IAA concentrations by an apparent feedback mechanism. The presence of exogenous auxins did not affect tryptophan labeling of either the endogenous tryptophan or IAA pools. This suggested that exogenous auxins did not alter the IAA biosynthetic pathway, but that synthetic auxins did appear to be necessary to induce callus proliferation, which was essential for excised hypocotyls to gain the competence to form somatic embryos.     

Low-fluence red light increases the transport and biosynthesis of auxin

In plants, light is an important environmental signal that induces photomorphogenesis and interacts with endogenous signals, including hormones. We found that light increased polar auxin transport in dark-grown Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum) hypocotyls. In tomato, this increase was induced by low-fluence red or blue light followed by 1 d of darkness. It was reduced in phyA, phyB1, and phyB2 tomato mutants and was reversed by far-red light applied immediately after the red or blue light exposure, suggesting that phytochrome is involved in this response. We further found that the free indole-3-acetic acid (IAA) level in hypocotyl regions below the hook was increased by red light, while the level of conjugated IAA was unchanged. Analysis of IAA synthesized from [13C]indole or [13C]tryptophan (Trp) revealed that both Trp-dependent and Trp-independent IAA biosynthesis were increased by low-fluence red light in the top section (meristem, cotyledons, and hook), and the Trp-independent pathway appears to become the primary route for IAA biosynthesis after red light exposure. IAA biosynthesis in tissues below the top section was not affected by red light, suggesting that the increase of free IAA in this region was due to increased transport of IAA from above. Our study provides a comprehensive view of light effects on the transport and biosynthesis of IAA, showing that red light increases both IAA biosynthesis in the top section and polar auxin transport in hypocotyls, leading to unchanged free IAA levels in the top section and increased free IAA levels in the lower hypocotyl regions.     

芦丁对绿豆幼苗营养生长的影响及其与IAA的相互作用

观察了植物体内的天然黄酮芦丁和吲哚乙酸(IAA)对绿豆幼苗营养生长的影响并测定胚轴中的芦丁和IAA含量.光照条件下芦丁(60μg/mL以下)处理对绿豆幼苗生长有一定促进作用,表现为胚轴和主根伸长加快、侧根数目增多、鲜重或干重增加;而光照条件下更高浓度芦丁(80μg/mL以上)处理及黑暗条件下芦丁(20～100μg/mL)处理对绿豆幼苗生长有抑制作用.当培养基中的芦丁浓度为60～80 μg/mL时,光照下的幼苗比暗处理的幼苗在胚轴中积累更多的芦丁;而芦丁浓度为40μg/mL以下和接近100μg/mL时幼苗在光照下累积的芦丁较暗处理的幼苗更少.0.1μg/mL以上的IAA促进芦丁的累积而进一步抑制幼苗胚轴和主根的伸长.当培养基中含有40 μg/mL的芦丁和0.5μg/mL的IAA时,胚轴中累积的芦丁达到高峰.芦丁降低黄化幼苗内源性IAA在胚轴中的累积,并抑制幼苗对IAA的吸收.     

Interaction of Brassinosteroids with Light Quality and Plant Hormones in Regulating Shoot Growth of Young Sunflower and <Emphasis Type="Italic">Arabidopsis</Emphasis> Seedlings

Sunflower hypocotyls elongate as light quality changes from the normal red to far-red (R/FR) ratio of sunlight to a lower R/FR ratio. This low R/FR ratio-induced elongation significantly increases endogenous concentrations of indole-3-acetic acid (IAA) and also of three gibberellins (GAs): GA₂₀, GA₁, and GA₈. Of these, it is likely GA₁ that drives low R/FR-induced growth. Brassinosteroids are also involved in shoot growth. Here we tested three R/FR ratios: high, normal, and low. Significant hypocotyl elongation occurred with this stepwise reduction in R/FR ratio, but endogenous castasterone concentrations in the hypocotyls remained unchanged. Brassinolide was also applied to the seedlings and significantly increased hypocotyl growth, though one that was uniform across all three R/FR ratios. Applied brassinolide increased hypocotyl elongation while significantly reducing (usually) levels of IAA, GA₂₀, and GA₈, but not that of GA₁, which remained constant. Given the above, we conclude that endogenous castasterone does not mediate the hypocotyl growth that is induced by enriching FR light, relative to R light. Similarly, we conclude that the hypocotyl growth that is induced by applied brassinolide does not result from an interaction of brassinolide with changes in light quality. The ability of applied brassinolide to influence IAA, GA₂₀, and GA₈ content, yet have no significant effect on GA₁, is hard to explain. One speculative hypothesis, though, could involve the brassinolide-induced reductions that occurred for endogenous IAA, given IAA’s known ability to differentially influence the expression levels of GA20ox, GA3ox, and GA2ox, key genes in GA biosynthesis.     

Changes in peroxidase and IAA oxidase activities during cell elongation in Phaseolus hypocotyls

Cytoplasmic and salt-extracted peroxidase and IAA oxidase activities were studied in Phaseolus vulgaris hypocotyls treated with gibberellic acid (GA, 200 μM), naphthyl acetic acid (NAA, 100 μM) and distilled water control (DW). Peroxidase activity was assayed with four hydrogen donors during the initial phase of hypocotyl elongation. Though peroxidase activity showed a decreasing trend with time in all the hydrogen donors studied; considerable variation with different hydrogen donors was observed. NAA had maximum peroxidase activity as compared to DW or GA treatment. The activity showed a clear inverse correlation with hypocotyl growth. IAA oxidase activity showed a similar trend with growth as peroxidase activity. A highly significant correlation was observed between peroxidase and IAA oxidase activities and high molecular weight xyloglucan content (P<0.001). Finally, the possible role of peroxidase and IAA oxidase activities in hypocotyl elongation growth is discussed.     

Changes in IAA,Phenolic Compounds,Peroxidase, IAA Oxidase,and Polyphenol Oxidase in Relation to Flower Formation in Crocus sativus

Changes in levels of IAA, phenolic compounds, peroxidase, polyphenol oxidase, and IAA oxidase activities in the corm and the apical bud of Crocus sativusL. during bud growth and development, with special emphasis on the flowering stage, were studied. In the bud, flower formation was accompanied by enhanced activities of peroxidase, polyphenol oxidase, IAA oxidase, and higher contents of phenolic compounds as well as lower levels of IAA. In the corm, during the flower formation, these enzymes showed an opposite behavior. Moreover, the contents of phenolics and IAA in the corm tissues during flower formation and growth were higher than at the other developmental stages. It may be concluded that the transition of saffron plants to flowering is correlated with peroxidase, polyphenol oxidase, and IAA oxidase. Furthermore, these enzymes might exert their roles in the regulation of flowering through their participation in IAA catabolism. The hypothesis of regulation of bud development by an interaction between phenolics and the enzymes involved in IAA catabolism is discussed.     

Phototropism in hypocotyls of radish. VI. No exchange of endogenous indole-3-acetic acid between peripheral and central cell layers during first and second positive phototropic curvatures

Using a physicochemical method, the distribution of endogenous indole-3-acetic acid (IAA) was measured in the peripheral and central cell layers, as well as in the illuminated and shaded sides of phototropically stimulated radish hypocotyls (Raphanus sativus var. hortensis f. gigantissimus Makino). The IAA was evenly distributed over the illuminated and shaded sides in the first and second positive phototropic curvatures induced by a pulse or a continuous unilateral illumination with blue light. Also, no net exchange of the IAA between the peripheral and central cell layers was observed during these curvatures. These results suggest that phototropism of radish hypocotyls cannot be described by the Cholodny-Went theory.     

The Relationship between Levels of Endogenous Gibberellins and the Response of Vigna Hypocotyls to Exogenous Indole-3-Acetic Acid

Segments excised from the upper and the lower parts of cowpea(Vigna unguiculata L.) hypocotyls were compared in terms oftheir responses to exogenous indole-3-acetic acid (IAA) in relationto their endogenous levels of gibberellin. Growth of the segmentswas measured continuously during xylem perfusion with a lineardifferential transformer. IAA induced a burst of elongationin the upper segments but only slight promotion of growth inthe lower segments. Treatment with uniconazole, a potent inhibitorof the biosynthesis of gibberellins, reduced the responsivenessof the upper segments to exogenous IAA to about one half ofthe control value. Pre-perfusion with GA₃ of such segments fortwo hours prior to application of IAA, partially restored theresponsiveness to IAA. Analysis by GC/MS identified GA₁, GA₄,GA₉ GA₂₀ and GA₅₁ as native gibberellins in the hypocotyls ofcowpea seedlings. Analysis by GC/SIM also showed that the physiologicallyactive gibberellins (GA₁ and GA₄) were located mainly in theupper part of the hypocotyl and the treatment with uniconazolemarketly reduced the endogenous level of gibberellins thereto less than 11% of the control level. These results suggestthat levels of endogenous gibberellins possibly control theresponse to IAA in these segments. (Received May 12, 1994; Accepted November 15, 1994)     

表油菜素内酯对黄瓜幼苗下胚轴过氧化物酶和IAA氧化酶活性的影响

黄瓜幼苗用0.1～1 ppm表油菜素内酯(epiBR)处理1～3d后,下胚轴中过氧化物酶活性明显低于对照;随着处理浓度的增加和处理时间的延长,与对照之间的差别愈趋增大。当浓度高于1ppm时,过氧化物酶的活性不再继续降低。表油菜素内醋对过氧化物酶活性的这种抑制作用需经约1O h的滞后期。IAA氧化酶的活性变化与过氧化物酶相似,epiBR处理时间愈长酶活性增加愈趋缓慢。 经IAA处理的下胚轴,过氧化物酶和IAA氧化酶的活性变化与对照无明显差异。这提示油菜素内酯与IAA促进生长可能是通过不同的作用方式。     

Influence of light conditions on development, apoplastic peroxidase activities and peroxidase isoenzymes in chicory root explants

Cichorium intybus L. (cv. Bea) root explants grown in continuous light had a higher fresh weight, lignin and chlorophyll content than explants grown in darkness. Intermediary values were found when the light conditions were switched after 6 days. Peroxidase activities (EC 1.11.1.7) were measured in the apoplastic fluid with guaiacol, indoleacetic acid (IAA) and syringaldazine as substrates. There was an inverse relationship between specific IAA oxidase activity and explant growth. Specific syringaldazine oxidase activity (SSO) correlated with the lignin content. Analysis by isoelectric focusing (IEF) showed that the apoplastic fluid contained both cationic and anionic peroxidase isoforms, whose expression differed according to culture conditions. Isoform isoelectric point (pI) 7.0 was only detectable when the explants were cultured for 12 days in darkness. When explants were grown for the first 6 days in light, SSO and lignin content were high and the isoforms pI 4.0, 6.6, 7.6 and 8.1 were highly expressed. Conversely, when the first 6 days were in darkness, specific IAA oxidase activity was high and the isoforms pI 4.5, 6.7 and 6.8. were most strongly expressed. The isoperoxidases pI 7.8 and 7.9 were strongly expressed when the explants were cultured for at least 6 days in darkness.     

The effect of 4-chlororesorcinol on the endogenous levels of IAA,ABA and oxidative enzymes in cuttings

Treatment of bean cuttings with 4-chlororesorcinol (4-CR), known to increase the number of roots and extend their distribution, prevented the accumulation of free indol-3-yl-acetic acid (IAA) in the hypocotyls within 24 h after cutting preparation. In mung bean there was no change in the distribution (upper half vs. 1 ower half of the hypocotyl) of IAA within the hypocotyl as a result of the treatment. In bean cuttings the treatment with 4-CR prevented the accumulation of IAA in the bottom of the cutting. Oxidation of IAA as a measure of IAA oxidase activity in bean was enhanced appreciably by 4-chlororesorcinol. The level of abscisic acid in mung bean, on the other hand, remained 3–4 fold higher than in the control, yet still about 50% lower than the zero time level. In untreated mung bean cuttings the activity of peroxidase increased after cutting preparation. In contrast, the activity of peroxidase in 4-Cr-treated cuttings was consistently lower. In order to relate to the effect of exogenously applied auxin the level of peroxidase was measured also in indol-3-yl-butyric acid-treated cuttings. The overall peroxidase activity in IBA-treated cuttings was not affected. However, when assaying for the different isozymes the drop in peroxidase activity was most evident in the inducible basic isoperoxidases both in 4-CR and IBA treatments. It appears that the exposure to 4-CR exerts an effect that is similar to that of exogenously applied auxin, affecting the activity of basic peroxidases and enhancing the oxidation of endogenous IAA, thus allowing the organization of the primordia.Abbreviations ABA - abscisic acid - 4-CR - 4-chlororesorcinol - IAA - indol-3-yl-acetic acid - IBA - indol-3-yl-butyric acid     

Adventitious rooting in hypocotyls of sunflower (Helianthus annuus) seedlings. IV. The role of changes in endogenous free and conjugated indole‐3‐acetic acid

We have studied the role of endogenous auxin on adventitious rooting in hypocotyls of derooted sunflower (Helianthus annuus L. var. Dahlgren 131) seedlings. Endogenous free and conjugated indole-3-acetic acid (IAA) were measured in three segments of hypocotyls of equal length (apical, middle, basal) by using gas chromatography-mass spectrometry with [¹³C₆]-IAA as an internal standard. At the time original roots were excised (0 h), the free IAA level in the hypocotyls showed an acropetally decreasing gradient, but conjugated IAA level increased acropetally; i.e. free to total IAA ratio was highest in the basal portion of hypocotyls. The basal portion is the region where most of root primordia were found. Some primordia were seen in this region within 24 h after the roots were excised. The quantity of free IAA in the middle portion of the hypocotyl increased up to 15 h after excision and then decreased. In this middle region there were fewer root primordia, and they could not be seen until 72 h. In the apical portion the amount of free IAA steadily increased and no root primordia were seen by 72 h. Surgical removal of various parts of the hypocotyl tissues caused adventitious root formation in the hypocotyl regions where basipetally transported IAA could accumulate. Reduction in the basipetal flow of auxin by N-1-naphthylphthalamic acid and 2,3,5-tri-iodobenzoic acid resulted in fewer adventitious roots. The fewest root primordia were seen if the major sources of endogenous auxin were removed by decapitation of the cotyledons and apical bud. Exogenous auxins promoted rooting and were able to completely overcome the inhibitory effect of 2,3,5-tri-iodobenzoic acid. Exogenous auxins were only partially able to overcome the inhibitory effect of decapitation. We conclude that in sunflower hypocotyls endogenously produced auxin is necessary for adventitious root formation. The higher concentrations of auxin in the basal portion may be partially responsible for that portion of the hypocotyl producing the greatest number of primordia. In addition to auxins, other factors such as wound ethylene and lowered cytokinin levels caused by excision of the original root system cuttings must also be important.