Effect of Ecdysterone on Morphological and Physiological Processes in Plants

In order to elucidate the physiological role of phytoecdysteroids in plants, we investigated the effects of exogenous ecdysterone (ECD) and phytohormones (IAA, GA₃, and 24-epibrassinolide (EBL)) on the growth of wheat coleoptiles and Arabidopsis thaliana seedlings (wild-type ecotype Columbia (Col) and its det2 mutant), on -amylase activity in the barley aleurone layer, and on the pigment content in the kidney bean senescent leaves. The range of effective ECD concentrations depended on the type of a reaction to be regulated. The regulation of growth processes was affected by a wide range of ECD concentrations (10^–13–10^–5 M), whereas some metabolic processes, such as the activation of -amylase and the retardation of leaf yellowing, by a narrow range, that is, 10^–9–10^–7 M and 10^–9–10^–8 M, respectively. We noted the synergetic effect of ECD and IAA on coleoptile elongation, the antagonistic effect of ECD and EBL on coleoptile elongation, as well as the antagonistic action of ECD and GA₃ on coleoptile elongation and -amylase activity. The data obtained demonstrate that ECD is a physiologically active compound. ECD might be supposed to act as a source of sterols or a regulator of IAA and protein synthesis. The effects of this regulator seems to be brought about by its interaction with the EBL and GA₃ receptors.     

The Effects of Indole-3-Acetic Acid and 2:4-Dichlorophenoxyacetic Acid on the Growth Rate and Endogenous Rhythm of Intact AvenaColeoptiles

The rates of elongation of the coleoptiles of Avena seedlings,subjected to intermittent immersion in solutions of IAA or 2:4-Dfor various total periods, were determined from measurementsof photographs taken every hour by infra-red radiation. Immersion in 17·5 mg./l. IAA for 1–5 hours causeda large increase in the growth rate followed by a depression.When the seedlings were immersed in 8·75 mg./l. IAA forperiods of 12 or 24 hours the depression was partially overcomeso long as the treatment was continued. Absorption of additionalIAA by the coleoptiles reduced their geotropic sensitivity. Penetration of 2:4-D (sodium salt) into the coleoptiles wasslower than that of IAA and the resulting stimulation of thegrowth rate was less, particularly in unbuffered solutions.After the treatment the growth rate declined slowly to aboutthe normal value. Results with coleoptiles were very similar to those previouslyobtained with rhizomes of Aegopodium and suggest that inhibitionof growth following stimulation by IAA may be of general occurrence.Possible causes of the inhibition are discussed and a comparisonis made between the results with intact coleoptiles and observationsmade by others on coleoptile sections. Temporary immersion of the seedlings in auxin solutions depressedthe rate of elongation of the primary leaf while it increasedthat of the coleoptile. It caused little disturbance of theendogenous rhythm induced by change from light to darkness.The suggestion that such rhythms can be explained in terms ofvariation in concentration of IAA-oxidase is not supported.     

The effect of chitooligosaccharides on hydrogen peroxide production and anionic peroxidase activity in wheat coleoptiles

We studied the effects of chitooligosaccharides (ChOS) with a mol wt of 5 kD, the degree of acetylation of 65%, and the concentrations from 0.01 to 100 mg/l on the content of hydrogen peroxide in incubation medium and the activity of anionic peroxidase (pI 3.5) in the segments of wheat (Triticum aestivum) coleoptiles. H₂O₂ production and peroxidase activity were found to be dependent on the ChOS concentration. After 3 h of incubation, the highest H₂O₂ level in medium was observed at 0.01 mg/l ChOS, whereas after 6h, at 1 mg/l. After 3 h of incubation, ChOS suppressed peroxidase activity. After 6 h of incubation, high ChOS concentrations enhanced peroxidase activity. IAA favored H₂O₂ accumulation in medium and suppressed anionic peroxidase. The involvement of ChOS in the control of the level of reactive oxygen species and anionic peroxidase activity in plant cells is suggested.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 238–242.Original Russian Text Copyright © 2005 by Yusupova, Akhmetova, Khairullin, Maksimov.This revised version was published online in April 2005 with a corrected cover date.     

Effects of the steroidal alkaloid tomatine in auxin bioassays and its interaction with indole-3-acetic acid

Summary The steroidal alkaloid tomatine did not enhance elongation of oat coleoptile and first internode sections, or of wheat coleoptile sections. Higher concentrations of the alkaloid inhibited elongation and interacted antagonistically with IAA. Although 10^-4 M tomatine alone did not influence elongation of oat coleoptile sections, it did reduce growth response to exogenous IAA. Tomatine concentrations less than 10^-4 M did not influence response to IAA. The auxin activity of tomatine, reported by Vendrig, was therefore not confirmed.     

Opposite effects of fusicoccin and IAA on putrescine synthesis of rice coleop tiles

Changes in polyamine biosynthesis and elongation of etiolated rice coleoptiles ( Oryza sativa L. cv. Taichung Native 1) in response to fusicoccin (FC) and indoleacetic acid (IAA) were investigated. FC stimulated coleoptile elongation at concentrations higher than 1 μ M but caused a decrease in the levels of free putrescine, spermidine and sper-mine, as well as in the activities of arginine decarboxylase (ADC, EC 4.1.1.19) and S -adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50). The extent to which FC caused these effects was dependent on its concentration. Treatment with 100 μ M IAA also induced coleoptile elongation and resulted in a decrease in free spermidine/sper-mine and SAMDC activity. However, treatment with IAA resulted in an increase in free putrescine levels and ADC activity. The extent of coleoptile elongation and putrescine accumulation also depended on IAA concentration. α-Difluoromethylarginine (DFMA), an irreversible inhibitor of ADC. but not α-difluoromethylornithine (DFMO). an irreversible inhibitor of ODC (EC 4.1.1.17), inhibited the LAA-stimulated coleoptile elongation and putrescine accumulation. Addition of putrescine could not reverse the effect of DFMA.     

Hexacyanoferrate (III) stimulation of elongation in coleoptile segments fromZea mays L.

Summary The influence of exogenous potassium hexacyanoferrate (III) (HCF III) on elongation of maize (Zea mays L.) coleoptile segments was investigated. Addition of HCF III led to a strong stimulation of growth both in the presence and absence of indole-3-acetic acid (IAA). The magnitude of growth stimulation was dependent on the presence of IAA, HCF III concentration, incubation time, and phase growth. The reduced form, potassium hexacyanoferrate (II), was without effect on growth. In the presence of HCF III, elongation was suppressed when coleoptile segments were treated with N,N-dicyclohexylcarbodiimide, cycloheximide or atebrine (quinacrine). The addition of HCF III stimulated the IAA-induced proton extrusion, and the e^–/H⁺ ratio decreased with incubation time. HCF III also strongly stimulated elongation ofAvena saliva L. coleoptile segments andGlycine max L. hypocotyl segments. These results suggested that a plasma membrane redox system (NADH oxidase type I) may be involved in the regulation of growth through the activity of the plasma membrane-bound ATPase.Abbreviations CH cycloheximide - DCCD N,N-dicyclohexylcarbodiimide - HCF III potassium hexacyanoferrate (III) (potassium ferricyanide) - HCF II potassium hexacyanoferrate (II) (potassium ferrocyanide) - IAA indole-3-acetic acid     

Inhibitory action of red light on the growth of the maize mesocotyl: evaluation of the auxin hypothesis

Brief irradiation of 3-d-old maize (Zea mays L.) seedlings with red light (R; 180 J m^-2) inhibits elongation of the mesocotyl (70–80% inhibition in 8 h) and reduces its indole-3-acetic acid (IAA) content. The reduction in IAA content, apparent within a few hours, is the result of a reduction in the supply of IAA from the coleoptile unit (which includes the shoot apex and primary leaves). The fluence-response relationship for the inhibition of mesocotyl growth by R and far-red light closely resemble those for the reduction of the IAA supply from the coleoptile. The relationship between the concentration of IAA (1–10 M) supplied to the cut surface of the mesocotyl of seedlings with their coleoptile removed and the growth increment of the mesocotyl, measured after 4 h, is linear. The hypothesis that R inhibits mesocotyl growth mainly by reducing the IAA supply from the coleoptile is supported. However, mesocotyl growth in seedlings from which the coleoptiles have been removed is also inhibited by R (about 25% inhibition in 8 h). This inhibition is not related to changes in the IAA level, and not relieved by applied IAA. In intact seedlings, this effect may also participate in the inhibition of mesocotyl growth by R. Inhibition of cell division by R, whose mechanism is not known, will also result in reduced mesocotyl elongation especially in the long term (e.g. 24 h).Abbreviations FR far-red light - IAA indole-3-acetic acid - P_fr phytochrome in the far-red-absorbing form - P_r phytochrome in the red-absorbing form - R red light     

The Relationship between the Growth of the Primary Leaf and of the Coleoptile in Seedlings of Avena and Triticum

Partial inhibition of extension growth of the primary leaf occurswhen whole Triticum seedlings are immersed in aerated solutionsof IAA but is replaced by growth promotion when sucrose is addedto the external solution. In seedlings in which the coleoptilehas been excised, IAA increases the growth of the leaf bothwith and without additional sucrose. Inhibition of the leaf by moderate concentrations of IAA nolonger occurs when the seedling is detached from the endosperm.Sucrose added to the external solution raised the percentageelongation of the coleoptile almost to the level of that attainedin intact seedlings without additional carbohydrate. It alsoenabled the leaf to show a positive growth response with IAA. The results indicate that in intact seedlings treated with IAAthe growth of the primary leaf is markedly diminished owingto diversion of carbohydrate to the coleoptile if the growthof the latter is promoted as a result of the treatment. Whenthe competition of the coleoptile for carbohydrate is diminishedor eliminated, acceleration of the growth of the primary leafby IAA becomes apparent. In addition to the endogenous rhythm, with a period close to24 hours, induced in the growth-rate of the coleoptile whenseedlings of Avena are transferred from red light to darkness,a similar rhythm, with a slightly longer period, is inducedin the growth-rate of the primary leaf. This rhythm persistsin elongating leaves so long as they remain within the coleoptile.It can be recorded for at least 100 hours in deseeded seedlings. When intact seedlings of Avena are immersed for one hour inrelatively high concentrations of IAA and then transferred todistilled water for 18 hours, the elongation of the coleoptileis greater and the inhibition of the leaf is less than whenthey are transferred to humid air. Sections of the leaf of Triticum showed a slight increase inelongation in concentrations of IAA up to 5 mg./l., but no evidencewas obtained that sections of leaf and coleoptile exert any.influenceon each other's elongation when floated together on solutionsof IAA.     

Comparison of the effect of some phenolic compounds on wheat coleoptile section growth with their effect on iaa-oxidase activity

P-coumaric acid (HCA), 2,4-dichlorophenol (DCP) and resorcionol acted as cofactors for IAA-oxidase isolated from young wheat plants. Ferulic acid (FA) and 3,4-dihydroxybenzoic acid (DHBA) induced a lag phase prior to IAA oxidation. HCA, FA (0.2-1 mg ml^-1) and DCP (0.03-1 mg ml^-1) strongly inhibited wheat coleoptile section growth. DHBA (0.01-1 mg ml^-1) slightly stimulated it and resorcinol was without effect. HCA inhibited IAA-induced growth of coleoptile sections and FA stimulated it at low IAA levels and inhibited it at higher ones. DHBA, DCP and resorcinol did not affect IAA-induced growth of coleoptile sections.     

The antagonism of IAA-induced hydrogen ion extrusion and coleoptile growth by diclofop-methyl

Diclofop-methyl (DM) (ester) was readily absorbed by peeled and unpeeled coleoptiles of wheat, Triticum aestivum L. cv. Waldron, and oat, Avena sativa L. cv. Garry. Substantial absorption of diclofop (acid) occurred only in peeled coleoptiles of the two species. IAA-induced acidification in peeled coleoptiles of both species was inhibited by 100 μ M DM or diclofop (acid) during a 3 to 4 h period. There was no recovery of acidification after DM or diclofop inhibition in oat coleoptiles; however, acidification in wheat coleoptiles recovered from inhibition by DM but not from diclofop. The recovery from DM inhibition may be due to a reduction in the diclofop pool derived from DM by efflux and metabolism (detoxification) in peeled wheat coleoptiles. Diclofop was not detoxified in oat coleoptiles. IAA-induced elongation of unpeeled oat coleoptiles was inhibited totally by 100 μ M DM but not by 100 μ M diclofop after 3.3 h of treatment. Wheat coleoptile elongation was relatively unaffected by either DM or diclofop. Basal elongation (no IAA) of both wheat and oat coleoptiles was inhibited by DM and diclofop. The inhibition by DM appeared to be irreversible, whereas the inhibition by diclofop was overcome by the addition of 10 μ M IAA.     

The pH profile for acid-induced elongation of coleoptile and epicotyl sections is consistent with the acid-growth theory

The acid-growth theory predicts that a solution with a pH identical to that of the apoplast of auxintreated tissues (4.5–5.0) should induce elongation at a rate comparable to that of auxin. Different pH profiles for elongation have been obtained, however, depending on the type of pretreatment between harvest of the sections and the start of the pH-incubations. To determine the acid sensitivity under in vivo conditions, oat (Avena sativa L.) coleoptile, maize (Zea mays L.) coleoptile and pea (Pisum sativum L.) epicotyl sections were abraded so that exogenous buffers could penetrate the free space, and placed in buffered solutions of pH 3.5–6.5 without any preincubation. The extension, without auxin, was measured over the first 3 h. Experiments conducted in three laboratories produced similar results. For all three species, sections placed in buffer without pretreatment elongated at least threefold faster at pH 5.0 than at 6.0 or 6.5, and the rate elongation at pH 5.0 was comparable to that induced by auxin. Pretreatment of abraded sections with pH-6.5 buffer or distilled water adjusted to pH 6.5 or above gave similar results. We conclude that the pH present in the apoplast of auxin-treated coleoptile and stems is sufficiently low to account for the initial growth response to auxin.Abbreviations FS free space - IAA indole-3-acetic acid This research was supported by a grant from the National Adonautics and space Administration (NASA), NAGW 1394 to R.E.C., NASA grant NAGW-297 to M.L.E., and NASA grant NAG 1849 to D.L.R.     

Comparison of the outer and inner epidermis : inhibition of auxin-induced elongation of maize coleoptiles by glucan antibodies

Polyclonal antibodies, raised against β-d-glucans prepared from oat (Avena sativa L.) caryopses, cross-reacted specifically with (1→3),(1→4)-β-d-glucans when challenged in a dot blot analysis of related polymers bound to a cellulose thin layer chromatography plate. The antibodies suppressed indoleacetic acid (IAA)-induced elongation of segments from maize (Zea mays L.) coleoptiles when the outer surface was abraded. However, IAA-induced elongation of nonabraded segments or segments with abrasion restricted to the interior of the cylinder was not influenced by the antibodies. Fab fragments prepared from the antibodies gave similar results. The capacity for IAA to overcome outward curvature of split coleoptile segments was partially reversed by treatment of the segments with the antibodies. Fluorescence microscopy revealed that antibody penetration was largely restricted to the epidermal cell wall region. These results support the view that the degradation of (1→3),(1→4)-β-d-glucans in the outer epidermal cell wall serves an essential role in auxin-induced elongation of Poaceae coleoptiles.     

表油菜素内酯促进小麦胚芽鞘伸长的作用

BR促进小麦胚芽鞘伸长的生理活性大于IAA,但高浓度的促进现象不如IAA明显。BR刺激乙烯生成与浓度相关。BR和IAA混合处理,对芽鞘切段的伸长、乙烯释放和H~+分泌都表现了加成作用。这二种激素在作用时间上有明显的差别,BR作用的滞后期更为清楚。BR有拮抗ABA对小麦胚芽鞘切段伸长的抑制作用。     

Rapid-growth responses of corn root segments: Effect of auxin on elongation

The effect of indole-3-acetic acid (IAA) on the elongation rates of 2 mm corn (Zea mays L.) root segments induced by citrate-phosphate buffer (or unbuffered) solutions of pH 4.0 and 7.0 was studied. At pH 7.0, auxin initially reduced the elongation rate in both buffered and unbuffered solutions. Only in buffer at pH 7.0 was auxin at a concentration of 0.1 M found to promote the elongation rate though briefly. THis promoted rate represented only ca. 20% of the rate achieved with only buffer at pH 4.0. Auxin in pH 4.0 buffered and unbuffered solutions only served to reduce the elongation rates of root segments. Some comparative experiments were done using 2 mm corn coleoptile segments. Auxin (pH 6.8) promoted the elongation rate of coleoptile segments to a level equal or greater than the maximal H ion-induced rate. The two responses of root segments to auxin are compared to auxin action in coleoptile growth.     

The effect of 5-azacytidine on the growth and thymidine kinase activity of coleoptile sections ofTriticum

The growth of coleoptile sections ofTriticum and its stimulation by indole-3-acetic acid (IAA) are inhibited by 5-azacytidine added into the cultivation medium. 50 per cent depression of the elongation was observed at 2×10^?3M 5-azacytidine concentration. Thymidine kinase activity in cell-free extracts prepared from coleoptile sections treated with 5-azacytidine, and caleulated per 10 mg of their wet weight, is increased while IAA administration resulted in its depression. The observed changes in thymidine kinase activity can be explained assuming the different uptake of water due to 5-azacytidine and IAA treatment.     

Effects of γ-irradiation on elongation and indole-3-acetic acid level of maize (Zea mays) coleoptiles

The effects of γ-irradiation on elongation and the level of indole-3-acetic acid (IAA) of maize (Zea mays) coleoptiles were investigated. When 3-day-old seedlings of maize were exposed to γ-radiation lower than 1 kGy, a temporal retardation of coleoptile elongation was induced. This retardation was at least partly ascribed to a temporal decrease in the amount of free IAA in coleoptile tips on the basis of the following facts: (1) the reactivity to IAA of the elongating coleoptile cells was not altered by irradiation; (2) endogenous IAA level in the tip of irradiated coleoptiles was at first unchanged, but then declined before returning to nearly the same level as that of the non-irradiated control; and (3) the amount of IAA that diffused from coleoptile tip sections showed a similar pattern to that of endogenous IAA. The rate of conversion between free and conjugated IAA was not significantly affected by irradiation. These results suggest that a temporal inhibition of maize coleoptile elongation induced by γ-irradiation can be ascribed to the reduction of endogenous IAA level in the coleoptile tip, and this may originate from the modulation in the rate of IAA biosynthesis or catabolism.     

Plant regeneration through callus initiation from thin mature embryo fragments of wheat

A wheat regeneration system was developed using mature embryos. Embryos were removed from surface-sterilised mature caryopses (winter wheat Odeon cultivar and spring wheat Minaret cultivar) and ground to pieces through a sterile nylon mesh. The fragments were characterised by means of the image analysis technique. They were 500 M mean diameter and most of them were elongated. They were used as explants to initiate embryogenic calli on solid medium supplemented with 10 M 2,4-dichlorophenoxyacetic acid. The morphogenic pathway of the initiated calli was followed for a 40-day culture period. Active cellular division occurred within 24 hours of cultivation. Several hundred calli were produced from 100 fragmented embryos within 3 days. A 90% callus induction rate was achieved and proembryos appeared by the 8th day of culture. The highest embryogenic calli induction rate of 47% was obtained when 2,4-dichlorophenoxyacetic acid was suppressed after a 3–4 week induction period. Two regeneration methods were finally compared. A total of 513 plantlets were produced. The optimal protocol produced 25–30 plants per 100 embryos. This regeneration method may be suitable for transformation applications.     

A study of the effects of fungitoxic compounds on Phytophthora cinnamomi in water

Copper and chlorine-releasing compounds were the most fungitoxic of 13 compounds tested in water for inhibition of Phytophthora cinnamomi. Mycelium was killed when immersed for 24 h in suspensions containing copper (13–45 mg/1) or a solution containing free residual chlorine (100 mg/1). Sub-lethal concentrations of these compounds reduced the numbers of sporangia. Exposing zoospores of P. cinnamomi for 60 s to water containing 2 mg free residual chlorine/1 reduced subsequent colony production on agar plates by 96–100%.
Prothiocarb, etridiazole ex. and furalaxyl killed mycelium immersed in solutions or suspensions for 3–6 days at 1500, 1000 and 600 mg a.i./l respectively and suppressed sporangium production at 1000, 500 and 300 mg/1.
Mycelium survived 3 days' immersion in ethyl hydrogen phosphonate compounds at 4000 mg a.i./l but 1000 mg a.i./l suppressed sporangium formation.
1-(2-Cyano-2-methoxyiminoacetyl)-3-ethyl urea and drazoxolon did not kill mycelium at 2000 and 1500 mg a.i./l respectively with a 6-day exposure, but reduced numbers of sporangia produced.     

Cooperation of Ethylene and Auxin in the Growth Regulation of Rice Coleoptile Segments

Elongation of coleoptile segments, having or not having a tip,excised from rice (Oryza sativa L. cv. Sasanishiki) seedlingswas promoted by exogenous ethylene above 0.3 µl l^–1as well as by IAA above 0.1 µM. Ethylene production ofdecapitated segments was stimulated by IAA above 1.0µM,and this was strongly inhibited by 1.0 µM AVG. AVG inhibitedthe IAA-stimulated elongation of the decapitated segment witha 4 h lag period, and this was completely recovered by ethyleneapplied at the concentration of 0.03 µl l^–1, whichhad no effect on elongation without exogenous IAA. The effectsof IAA and ethylene on elongation were additive. These factsshow that ethylene produced in response to IAA promotes ricecoleoptile elongation in concert with IAA, probably by prolongingthe possible duration of the IAA-stimulated elongation, butthat they act independently of each other. Moreover, AVG stronglyinhibited the endogenous growth of coleoptile segments withtips and this effect was nullified by the exogenous applicationof 0.03 µl l^–1 ethylene. These data imply that theelongation of intact rice coleoptiles may be regulated cooperativelyby endogenous ethylene and auxin in the same manner as foundin the IAA-stimulated elongation of the decapitated coleoptilesegments. Key words: oryza sativa, Ethylene, Auxin, Coleoptile growth