The Role of Various Regions of the Bean Hypocotyl on Red Light-induced Hook Opening

Measurement of various zones on the concave half of etiolated Phaseolus vulgaris L. (cv. Black Valentine) hypocotyls has shown that growth at the basal portion of the elbow and the contiguous upper portion of the shank was stimulated earliest by red light. Growth of these two zones was unaffected by the tissue of the convex half but was inhibited by tissue distal to them. The inhibition was alleviated by the continuous presence of shank tissue below the growing zones. Based on cuts made halfway through the hypocotyl at positions above, below, or between the two zones of growth, it is suggested that cells at the inner portion of the upper shank control in some way the light-induced growth of the elbow cells directly above.     

Factors involved in the opening of the hypocotyl hook of cotton and beans

Conditions influencing the opening of the bean (Phaseolus vulgaris L.) and cotton (Gossypium hirsutum L.) hypocotyl hook were defined. Such hooks were shown to undergo geotropic curvature; orientation of the hook with respect to gravity greatly affected the observed opening. Cotton and bean hooks behaved exactly opposite in regard to the presence of the cotyledons and apical bud. The cotton hook required the cotyledons for opening, but the corresponding tissue slowed or inhibited opening of the bean hook. With cotton, lower hypocotyl and root tissues stimulated hook opening, but with bean, the tissues below the hook section had little effect. Kinetin and gibberellic acid both modified hook opening in light and dark; the former was inhibitory and the latter was stimulatory. Indoleacetic acid, at concentrations above 10⁻⁵ M, caused pronounced hook closing in red light but not in the dark. These effects were generally the same with both plants. In opening of the cotton hook, the cotyledons were not necessary as a light receptor tissue. None of the growth substances tested were able to substitute completely for the cotton cotyledon. Coumarin was a pronounced inhibitor of opening of the cotton hook, and this response was expressed to such a degree as to cause hook closure with bean tissue. Reduced oxygen levels inhibited hook opening in bean. Oxygen was required in processes subsequent to the light reaction, but not for the photochemical process.     

Dim-Red-Light-Induced Increase in Polar Auxin Transport in Cucumber Seedlings : I. Development of Altered Capacity, Velocity, and Response to Inhibitors

We have developed and characterized a system to analyze light effects on auxin transport independent of photosynthetic effects. Polar transport of [³H]indole-3-acetic acid through hypocotyl segments from etiolated cucumber (Cucumis sativus L.) seedlings was increased in seedlings grown in dim-red light (DRL) (0.5 μmol m⁻² s⁻¹) relative to seedlings grown in darkness. Both transport velocity and transport intensity (export rate) were increased by at least a factor of 2. Tissue formed in DRL completely acquired the higher transport capacity within 50 h, but tissue already differentiated in darkness acquired only a partial increase in transport capacity within 50 h of DRL, indicating a developmental window for light induction of commitment to changes in auxin transport. This light-induced change probably manifests itself by alteration of function of the auxin efflux carrier, as revealed using specific transport inhibitors. Relative to dark controls, DRL-grown seedlings were differentially less sensitive to two inhibitors of polar auxin transport, N-(naphth-1-yl) phthalamic acid and 2,3,5-triiodobenzoic acid. On the basis of these data, we propose that the auxin efflux carrier is a key target of light regulation during photomorphogenesis.     

Far red light control of elongation in light-grown bean hypocotyl: Effect on different age zones and interaction with indole-3-acetic acid

The effect of far red light on the light-grown bean hypocotyland its interaction with indole-3-acetic acid (IAA) were studied.Elongation of younger zones of the hypocotyl was inhibited butthat of older zones was promoted by far red light. This wascontrolled by phytochrome. Both the hook and shank portionscould receive far red light and its effect could be transmittedto either portions of the hypocotyl. When IAA was applied to the upper cut surface of the hypocotylunit, elongation of the shank portion was promoted even withoutfar red irradiation. IAA did not change the aspect of the growthcurves but amplified the elongation of each zone. When IAA wasapplied to each zone of the shank portion, elongation of zonesolder than the treated one was promoted but that of youngerzones was inhibited. This effect was emphasized by far red light.When IAA was applied to the older shank portion, elongationof the treated zone was synergistically promoted by IAA andfar red light, but when applied to the elbow or younger shankportion, far red light completely nullified the promoting effectof IAA. (Received October 1, 1979; )     

A role for ABCB19‐mediated polar auxin transport in seedling photomorphogenesis mediated by cryptochrome 1 and phytochrome B

During seedling establishment, blue and red light suppress hypocotyl growth through the cryptochrome 1 (cry1) and phytochrome B (phyB) photosensory pathways, respectively. How these photosensory pathways integrate with growth control mechanisms to achieve the appropriate degree of stem elongation was investigated by combining cry1 and phyB photoreceptor mutations with genetic manipulations of a multidrug resistance‐like membrane protein known as ABCB19 that influenced auxin distribution within the plant, as evidenced by a combination of reporter gene assays and direct auxin measurements. Auxin signaling and ABCB19 protein levels, hypocotyl growth rates, and apical hook opening were measured in mutant and wild‐type seedlings exposed to a range of red and blue light conditions. Ectopic/overexpression of ABCB19 (B19OE) greatly increased auxin in the hypocotyl, which reduced the sensitivity of hypocotyl growth specifically to blue light in long‐term assays and red light in high‐resolution, short‐term assays. Loss of ABCB19 partially suppressed the cry1 hypocotyl growth phenotype in blue light. Hypocotyl growth of B19OE seedlings in red light was very similar to phyB mutants. Altered auxin distribution in B19OE seedlings also affected the opening of the apical hook. The cry1 and phyB photoreceptor mutations both increased ABCB19 protein levels at the plasma membrane, as measured by confocal microscopy. The B19OE plant proved to be a useful tool for determining aspects of the mechanism by which light, acting through cry1 or phyB, influences the auxin transport process to control hypocotyl growth during de‐etiolation.     

The role of light and growth regulators in the opening of the dentaria petiolar hook

The phenomenon of the etiolated hook is not restricted to the hypocotyl of the dicotyledenous plant (e.g., Phaseolus) but appears to serve a similar, adaptive function in the petioles of certain rhizomatous plants. The commonly employed regulants of hypocotyl hook opening were tested for their effect on the petiolar hook of Dentaria diphylla. The hook was found to require both light (red light promoted, far red inhibited) and the intact leaf for opening. The leaf requirement was fully replaced by gibberellic acid (0.04% in lanolin) but only in light; cobalt chloride (0.1-1.0 mm) promoted a partial opening in dark with or without leaf; and coumarin (1 mm), indoleacetic acid (1-4% in lanolin), and ethylene 10 microliter per liter all inhibited opening of hooks with or without lamina. The absolute requirement for light and leaf tissue and the replacement of proximal tissue by GA₃ alone represent marked differences in the physiology of hypocotyl and petiolar hooks. These differences are believed to indicate the necessity for concomitant leaf maturation in petiolar hook opening.     

Gene expression profile of zeitlupe/lov kelch protein1 T-DNA insertion mutants in Arabidopsis thaliana: Downregulation of auxin-inducible genes in hypocotyls

Elongation of hypocotyl cells has been studied as a model for elucidating the contribution of cellular expansion to plant organ growth. ZEITLUPE (ZTL) or LOV KELCH PROTEIN1 (LKP1) is a positive regulator of warmth-induced hypocotyl elongation under white light in Arabidopsis, although the molecular mechanisms by which it promotes hypocotyl cell elongation remain unknown. Microarray analysis showed that 134 genes were upregulated and 204 genes including 15 auxin-inducible genes were downregulated in the seedlings of 2 ztl T-DNA insertion mutants grown under warm conditions with continuous white light. Application of a polar auxin transport inhibitor, an auxin antagonist or an auxin biosynthesis inhibitor inhibited hypocotyl elongation of control seedlings to the level observed with the ztl mutant. Our data suggest the involvement of auxin and auxin-inducible genes in ZTL-mediated hypocotyl elongation.     

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.     

The Arabidopsis mutant alh1 illustrates a cross talk between ethylene and auxin

Ethylene or its precursor 1-aminocyclopropane-1-carboxylic acid (ACC) can stimulate hypocotyl elongation in light-grown Arabidopsis seedlings. A mutant, designated ACC-related long hypocotyl 1 (alh1), that displayed a long hypocotyl in the light in the absence of the hormone was characterized. Etiolated alh1 seedlings overproduced ethylene and had an exaggerated apical hook and a thicker hypocotyl, although no difference in hypocotyl length was observed when compared with wild type. Alh1 plants were less sensitive to ethylene, as reflected by reduction of ACC-mediated inhibition of hypocotyl growth in the dark and delay in flowering and leaf senescence. Alh1 also had an altered response to auxin, whereas auxin levels in whole alh1 seedlings remained unaffected. In contrast to wild type, alh1 seedlings showed a limited hypocotyl elongation when treated with indole-3-acetic acid. Alh1 roots had a faster response to gravity. Furthermore, the hypocotyl elongation of alh1 and of ACC-treated wild type was reverted by auxin transport inhibitors. In addition, auxin up-regulated genes were ectopically expressed in hypocotyls upon ACC treatment, suggesting that the ethylene response is mediated by auxins. Together, these data indicate that alh1 is altered in the cross talk between ethylene and auxins, probably at the level of auxin transport.     

Ethylene and carbon dioxide as mediators in the response of the bean hypocotyl hook to light and auxins

Summary Ethylene inhibits hook opening in the bean hypocotyl and at high concentrations induces closure of the hook. Indoleacetic acid and 2,4-dichlorophenoxyacetic acid, whose inhibitory effect on hook opening resembles that of ethylene, stimulate ethylene production from the hook tissue, and this ethylene production is physiologically active in inhibiting hook opening. It is concluded that the inhibition of opening by auxin is due at least in a major part to auxin-induced ethylene production by the hook tissue.Carbon dioxide promotes hook opening, apparently by antagonizing the action of endogenous ethylene. The concentration of respiratory CO₂ in the internal gas space of the hook tissue is high enough to play a role in the regulation of hook opening.Red light causes a decrease in ethylene production and an increase in CO₂ evolution from the hook tissue. These effects are partially reversible by far-red light. It is concluded that both ethylene and CO₂ serve as natural growth regulators which mediate the hypocotyl hook-opening response to light in bean seedlings.     

Identification of dehydrocostus lactone and 4-hydroxy-β-thujone as auxin polar transport inhibitors

The survey of naturally occurring of auxin polar transport regulators in Asteraceae was investigated using the radish (Raphanus sativus L.) hypocotyl bioassay established in this study. Significant auxin polar transport was observed when radiolabeled indole-3-acetic acid (IAA) was applied at the apical side of radish hypocotyl segments, but not when it was applied at the basal side of the segments. Almost no auxin polar transport was observed in radish hypocotyl segments treated with synthetic auxin polar transport inhibitors of N-(1-naphthyl)phthalamic acid (NPA) and 9-hydroxyfluorene-9-carboxylic acid (HFCA) at 0.5 μg/plant. 2,3,5-Triiodobenzoic acid (TIBA) at 0.5 μg/plant was less effective than NPA and HFCA, and p-chlorophenoxyisobutyric acid (PCIB) at 0.5 μg/plant had almost no effect on auxin polar transport in the radish hypocotyl bioassay. These results strongly suggest that the radish hypocotyl bioassay is suitable for the detection of bioassay-derived auxin polar transport regulators. Using the radish hypocotyl bioassay and physicochemical analyses, dehydrocostus lactone (decahydro-3,6,9-tris-methylene-azulenol(4,5-b)furan-2(3H)-one) and 4-hydroxy-β-thujone (4-hydroxy-4-methyl-1-(1-methylethyl)-bicyclo[3.1.0]hexan-3-one) were successfully identified as auxin polar transport inhibitors from Saussurea costus and Arctium lappa, and Artemisia absinthium, respectively. About 50 and 40 % inhibitions of auxin polar transport in radish hypocotyl segments were observed at 2.5 μg/plant pre-treatment (see “Materials and methods”) of dehydrocostus lactone and 4-hydroxy-β-thujone, respectively. Although the mode of action of these compounds in inhibiting auxin polar transport has not been clear yet, their possible mechanisms are discussed.     

Involvement of Ethylene in Gravity-Regulated Peg Development in Cucumber Seedling

Peg development on the lower side of the transition (TR) zoneof the hypocotyl and the root in cucumber seedlings was inhibitedby two inhibitors of ethylene biosynthesis, aminoethoxyvinylglycine and aminooxyacetic acid, and by an inhibitor of ethyleneaction, Ag-thiosulfate. These ethylene inhibitors also inhibitedplumular hook formation of the cucumber hypocotyl. When cucumberseeds were germinated in a vertical position or on a horizontalclinostat, the seedlings exhibited straight growth without formationof a plumular hook and failed to develop a protuberant peg.In the seedlings germinated in a vertical position, exogenousIAA induced a distinct peg-like protuberance, whereas ACC andethylene stimulated overall swelling around the TR zone, whichobviously differed from the normal peg. In horizontally placedseedlings, however, peg development was more pronounced dueto treatment with 5µl/liter of ethylene. These resultsindicate that a high ethylene level in the hook region playssome role in peg development. TIBA, an inhibitor of auxin transport,at 10^–4 M inhibited peg development, as reported previouslyby Witztum and Gersani (1975), but a somewhat lower concentrationof TIBA induced two distinct pegs, on both the lower and uppersides of horizontally grown seedlings. (Received June 12, 1987; Accepted December 11, 1987)     

Control of Hypocotyl Hook Angle in Phaseolus mungo L. Role of Growth Substances

Effects of growth hormones on the hook angle and light responseof Phaseolus mungo L. hypocotyl hooks are described and theresults are discussed with reference to the functions of otherparts of the seedling in controlling the growth and shape ofthe hook. Apically applied IAA (indolyl acetic acid) prevented hook openingin decapitated seedlings in the dark and in all the red-irradiatedseedlings. [¹⁴C]IAA experiments showed that only a small quantityof IAA (2–6 ?g per hook) was required to produce theseeffects, and that transport of IAA through the hook was negligibleand unaffected by red irradiation. ABA (abscisic acid) had little effect on the hooks or theirlight response. Applied ethylene and IAA-induced ethylene slightly closed thehooks, but only slightly reduced light-induced opening. IAAreduced the effect of ethylene in the dark, but after irradiationthe hooks appeared more sensitive to the ethylene in the presenceof IAA, resulting in light-induced hook closure. Basally applied kinetin (6-furfurylaminopurine) prevented decapitatedhooks from opening in the dark, especially when GA₃ (gibberellicacid) was also present. Some combinations of kinetin and GA₃(with high kinetin concentrations) also prevented light-inducedopening, but combinations with lower kinetin concentrationsallowed almost as much opening as was found in intact hooks. It is proposed that the terminal parts act by regulating thesupply of cytokinins and gibberellins from the basal parts,and that IAA does not mediate this funotion in this species. The results are compared with those reported for other species.     

Role of growth regulators in the bean hypocotyl hook opening response

Summary The opening of the hypocotyl hook in bean seedlings is due to a rapid elongation of cells on the inner side of the hook elbow. Red light promotes hook opening by inducing this cell elongation.Opening is inhibited by low concentrations of indoleacetic acid (IAA) and 2,4-dichlorophenoxyacetic acid (2,4-D), and higher concentrations of these auxins cause a closure of the hook. In darkness, opening is induced slightly by p-chlorophenoxyisobutyric acid (PCIB), whereas in red light this auxin antagonist promotes opening only when IAA is added simultaneously to inhibit opening.The amount of diffusible auxin released by the hook tissue is not affected by red illumination that is sufficient to induce maximal hook opening.Gibberellic acid (GA) promotes the hook opening. The magnitude of its effect is, however, rather small, especially in darkness. (2-Chloroethyl)-trimethylammonium chloride (CCC) and 2-isopropyl-4-(trimethylammonium-chloride)-5-methylphenyl piperidine-1-carboxylate (Amo-1618) inhibit hook opening in red light, and this inhibition is completely overcome by addition of GA.Cytokinins and abscisic acid at rather high concentrations inhibit hook opening in light but produce no significant effect in darkness.Hook opening is promoted by Ca⁺⁺ and K⁺, and notably by Co⁺⁺ and Ni⁺⁺.It is concluded that 1. endogenous gibberellin assists in hook opening, but light does not act by changing the gibberellin level; 2. light does not act by decreasing the endogenous auxin level; and 3. cytokinins or abscisic acid do not seem to have a special role in the response.     

The Influence of Auxin and Ethylene on Chromatin-directed Ribonucleic Acid Synthesis in Soybean Hypocotyl

Soybean seedlings treated with ethylene exhibited small increases in ribonucleic acid content in the elongating section of the hypocotyl. Chromatin isolated from the elongating section of ethylene-treated seedlings showed a 35 to 60% increase in the capacity for RNA synthesis. The ethylene-induced response was saturated at 1 microliter/liter of ethylene and was fully expressed after 3 hours. Auxin caused marked accumulation of RNA and DNA in the elongating and basal tissue of the hypocotyl. Chromatin isolated from these auxin-treated tissues showed an 8- to 10- fold increase in RNA synthetic capacity as measured in vitro. Ethylene added with auxin reduced the auxin enhancement of nucleic acid synthesis in the elongating and basal tissues. Both ethylene and auxin treatment of the seedlings inhibited nucleic acid accumulation and chromatin activity in the apical tissue. Ethylene did not appear to mediate the auxin effects on nucleic acid synthesis in soybean hypocotyl with the possible exception of inhibition in the apical tissue.     

Evidence for a Relationship between H Excretion and Auxin in Shoot Gravitropism

The role of auxin and protons in the gravitropic response of the sunflower (Helianthus annuus L. cv Sungold) hypocotyl has been investigated. No physiological asymmetry in acid-growth capacity could be detected between the upper and lower surfaces of gravistimulated hypocotyls. These data imply that neutral buffers inhibit shoot gravitropism by preventing the establishment of a lateral proton gradient along gravitropically stimulated hypocotyls. Indirect evidence that auxin is involved in the establishment and/or maintenance of such a gradient derives from the quantitative assessment of the effects of exogenous auxin, anti-auxins, and vanadate on gravicurvature. At low concentrations, exogenous auxin accelerated curvature; at high concentrations, curvature was prevented. Vanadate, an inhibitor of auxin-enhanced H⁺ secretion, α-(p-chlorophenoxy)isobutyric acid (PCIB), an anti-auxin, and 2,3,5-triiodobenzoic acid (TIBA), an auxin-transport inhibitor, prevented observable asymmetric proton excretion using a brom cresol purple agar technique and also inhibited gravicurvature. Vanadate, PCIB, and TIBA inhibition of gravicurvature could be reversed with acid treatment to the lower surface of a gravistimulated hypocotyl. Auxin treatment to the lower surface of a gravistimulated hypocotyl did not reverse vanadate-induced inhibition, but it did partially reverse PCIB- and TIBA-induced inhibition. These results indicate a close relationship between the acid-growth theory and the differential growth responses of the sunflower hypocotyl during gravitropism.     

The positive hook: the role of gravity in the formation and opening of the apical hook

Photographic observations on germinating seedlings of Lepidium sativum L., Cucumis sativus L., and Helianthus annuus L. showed that the hypocotyl hook is not present in the seed but forms during the early stages of growth. Evidence that gravity plays a major role in inducing curvature of the hypocotyl, and in maintaining the hook once it has been formed, was obtained from clinostat experiments, from the use of morphactin to remove geotropic sensitivity and from inversion of seedlings to change the direction of the geostimulus. In L. sativum and H. annuus gravity perception seemed to be the only mechanism responsible for hook formation. In C. sativus hook formation was additionally aided by the mode of emergence of the cotyledons from the seed coat but gravity played an indirect role in regulating such emergence. Further evidence that hook formation is linked to a georesponse was derived from a comparison of hypocotyl development in wild-type Arabidopsis thaliana seedlings with that of an ageotropic mutant, hook formation being found to occur only in the wild type. Hook formation and maintenance is discussed in terms of contrasting geosensitivity between the apical and basal ends of the hypocotyl and it is suggested that light-induced hook opening is a reversal to a condition of uniformly negative georesponse throughout the hypocotyl.     

Involvement of ethylene in responses of etiolated bean hypocotyl hook to coumarin

Coumarin, at concentrations between 1.0 and 0.1 mm, inhibited red light-induced opening of the etiolated bean hypocotyl hook. In addition, anthocyanin synthesis and geotropic bending were inhibited. Coumarin stimulated ethylene synthesis, and ethylene was shown to mediate the inhibitory actions of coumarin. This conclusion was supported by: (a) the parallel concentration dependence and time sequence of hook closing and ethylene synthesis, (b) the restriction of the bulk of coumarin-induced ethylene production to the curved portion of the hook where opening is expressed, (c) the ability of both coumarin and ethylene to reclose partially opened hooks, and (d) the ability of exogenous ethylene, in the amounts produced by coumarintreated hooks, to duplicate the inhibitory effects of coumarin. There was an increasing stimulation of growth of the straight portion of the hypocotyl hook section as coumarin concentrations were increased from 0.1 to 1.0 mm. This action of coumarin was not duplicated by ethylene and occurred regardless of the presence or absence of added ethylene. The results of this study suggest that many actions of coumarin in growth systems are mediated by ethylene produced in response to the coumarin.     

Effects of Brassin-Complex on Auxin and Gibberellin Mediated Events in the Morphogenesis of the Etiolated Bean Hypocotyl

The excised, hooked bean hypocotyl was the system used to determine wheiher the ‘auxin- and gibberellin like’ effect of the lipoidal pollen extract, Brass in-complex (Br), were mediated through, or independent of, auxin and gibberellin. The morphogenetic events of hook opening and hypocotyl elongation in this system are regulated by auxin and gibberellin, respectively. Brassin complex, like IAA, elicited a book closure in (he dark and retarded its opening in red light. This effect was synergized by T1BA, IAA and the presence of the auxin-producing organs, the epicotyl and cotyledons. Br-elicited hook closure was inhibited by the antiauxin. PCIB. Both GA₃ and Br totally reversed the light inhibition of hypocotyl elongation. The GA₃-effect, but nol the Br elicited elongation, was overcome by Ancymidol. Hypocotyl elongation was partially inhibited by TIBA and PCIB. suggesting a possible auxin involvement also in this effect of Br. Br may elicit its growth responses through an effect on endogenous auxin levels, In this way it is different from other lipoidat growth regulators, such as the oleanimins which require the presence of exogenous growth regulators for activity.     

Interactions of light and ethylene in hypocotyl hook maintenance in Arabidopsis thaliana seedlings

Etiolated seedlings frequently display a hypocotyl or epicotyl hook which opens on exposure to light. Ethylene has been shown to be necessary for maintenance of the hook in a number of plants in darkness. We investigated the interaction of ethylene and light in the regulation of hypocotyl hook opening in Arabidopsis thaliana . We found that hooks of Arabidopsis open in response to continuous red, far-red or blue light in the presence of up to 100 μl l⁻¹ ethylene. Thus a change in sensitivity to ethylene is likely to be responsible for hook opening in Arabidopsis, rather than a decrease in ethylene production in hook tissues. We used photomorphogenic mutants of Arabidopsis to demonstrate the involvement of both blue light and phytochrome photosensory systems in light-induced hook opening in the presence of ethylene. In addition we used ethylene mutants and inhibitors of ethylene action to investigate the role of ethylene in hook maintenance in seedlings grown in light and darkness.