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.     

Effect of the Hypocotyl Hook on Chlorophyll Accumulation in Excised Cotyledons of Cucumis sativus L

Hypocotyl hooks have been shown to influence greening in excised cucumber (Cucumis sativus) cotyledons. The properties of the lag phase are greatly affected by the presence or absence of the hook tissue. A 45-second light pretreatment followed by 4 hours of darkness is sufficient to remove the lag phase from cotyledons with hooks, while hookless cotyledons require 2 hours of continuous illumination followed by 1 hour of dark incubation to break the lag phase. The effect of hooks on cotyledon greening is enhanced if the hooks are shielded from light. Cutting off the hooks after lag phase removal caused a marked decrease in chlorophyll accumulation in the cotyledons. These observations may indicate that the hypocotyl hooks produce a substance or substances needed in the greening process, which are translocated to the cotyledons. Indoleacetic acid, abscisic acid, gibberellin A₃, 6-benzylamino purine and δ-aminolevulinic acid do not show any activity; on the other hand, ethylene appears to replace partially the hypocotyl hooks.     

Opening of the hypocotyl hook in seedlings as influenced by light and adjacent tissues

The influence of the cotyledons and apical bud and the root system on the light-induced opening of the hypocotyl hook of etiolated seedlings of Gossypium hirsutum L., Phaseolus vulgaris L., Helianthus annuus L., Ipomoea alla L., Ipomoea sp., Cucumis sativus L., Linum usitatissimum L., Hibiscus esculentus L., and Raphanus sativus L. was studied. Light stimulated the opening of hypocotyl hook in all cases, but the cotyledons and roots had different effects in different plants. Generally, the presence of cotyledons and the remainder of the shoot apical to the hook inhibited light-mediated opening, but in Gossypium the organs stimulated light-mediated opening. Presence of roots either promoted opening, had no effect, or had an effect only when the cotyledons were present. In the dark the adjacent organs had a reduced effect over that shown in the light, but one cultivar of cotton, Acala SJ1, opened the hook in the dark without cotyledons as much as under any condition in the light. The variation between species in hook opening may related to the need of that process for a proper hormonal balance, as affected by light, which must be obtained from adjacent tissues.     

Attempts to demonstrate incorporation of labelled precursors into acetylcholine by Phaseolus vulgaris seedlings

The labelling of acetylcholine was investigated in bean seedlings (Phaseolus vulgaris) and hypocotyl hooks. Labelling could only be found after red light irradiation but not in the dark. Acetate was a much better precursor than choline. Glucose gave no detectable amounts of acetylcholine labelling during 24 hr of incubation. The rate of acetylcholine labelling was higher in whole seedlings than in hypocotyl hook preparations.     

A double enhancement of greening in the cotyledons of cucumber seedlings by excision and red light

Cotyledons excised without the hypocotyl hook from 6-day-old etiolated cucumber ( Cucumis sativus L. var. Elem) seedlings accumulated a significantly higher amount of chlorophyll than cotyledons excised with hooks or intact cotyledons. It was found that maximum ehancement of greening was achieved after 2 h of dark incubation following excision. Pretreatments with red light effected an additive rise in chlorophyll level in subsequent white light after a dark incubation, suggesting that the effects of excision and phytochrome on greening act independently. Etiolated seedlings were variously dissected before greening and it was found that enhancement occurred only when cotyledons were excised at the level of the hypocotyl hook or above it. Similar results were obtained when the dissected plants were pre-treated with red light.     

Effect of the hypocotyl hook on greening in etiolated cucumber cotyledons

Four-day-old etiolated cucumber cotyledons (Cucumis sativus, L.) were excised and allowed to green in white fluorescent light at 28 C. Cotyledons excised with a full hypocotyl hook exhibited a lag phase of 1 hour before entering the rapid greening phase, whereas cotyledons excised without any hypocotyl hook exhibited a lag phase of 6 hours. Cotyledons excised with varying lengths of hypocotyl hook accumulated chlorophyll roughly in proportion to the hook length. When cotyledons were excised with a full hook and were partially or totally shielded from light with aluminum foil, the samples with the hook covered accumulated more chlorophyll than the wholly exposed samples. The samples with the cotyledons covered showed no net accumulation of chlorophyll irrespective of hook's exposure to light. These data suggest the contribution of some factor or factors by the hypocotyl hook which reduce the lag phase during greening.     

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.     

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.     

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.     

Control of the Hypocotyl Hook Angle in Phaseolus mungo L.: the Role of Parts of the Seedling

Phaseolus mungo L. seedlings were grown in the dark for 5 d.They were either kept in the dark or exposed to 5 min red light.The location of the light-sensitive region (the hook) and theanatomy of light-induced opening are described. The responsewas phytochrome-mediated. The time course of light-induced openingand its far-red reversal are described and the implicationsfor the mechanism of light-induced opening discussed. The lagphase between irradiation and the start of opening was >8h, for 3 h of which the response remained completely photoreversible.This implies that some critical level of a factor is requiredbefore hook opening can be initiated. The terminal parts (leavesand cotyledons) kept the hooks shut in the dark, but did notdirectly affect the light response. The necessity for distinguishingbetween treatment effects on hook angle and on light-inducedopening is noted. We hypothesize that the terminal parts produceda hook-maintaining factor whose level declined after their removal.This factor was dispersed or inactivated following irradiation,or the hook cells were rendered less sensitive to it. Differencesbetween the responses of P. mungo and those reported for otherspecies are discussed.     

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 inhibitors of RNA and protein synthesis on bean hypocotyl hook opening and their implications regarding phytochrome action

Summary Inhibitors of protein and RNA synthesis (cycloheximide, puromycin, chloramphenicol, and actinomycin D), as well as Co⁺⁺, induce opening of the hypocotyl hook of bean seedlings during the early stage of the opening period both in the darkness and red light. The response is transitory, however, complete straightening of a hook can not be achieved in the presence of these agents. These agents abolish the response of hooks to red illumination. They also block the suppression of hook opening caused by IAA and ethylene. The response and sensitivity to GA are not affected by the inhibitors. Inhibitors of DNA synthesis (FUDR and mitomycin C) have no effect on hook opening. It appears that in this growth response RNA and protein synthesis are more immediately involved in ethylene action than they are in the cell elongation process or the action of GA thereon.The results indicate that phytochrome does not induce hook opening simply by activating genes whose products directly promote growth. It is suggested that the regulation of ethylene formation by light and auxins may be exerted by way of influences on tissue levels of phenolic inhibitors of ethylene biosynthesis.     

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.     

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.     

Influence of Light on the Bioelectric Potential of the Bean (Phaseolus vulgaris) Hypocotyl Hook

By use of surface electrodes electropotenlial measurements were carried out on hypocotyl hooks of Phaseolus vulgaris seedlings. The hooks were illuminated with a small spot of white, blue, red or far red light. The potential changes in bean hypocotyl hooks do not show the red-far red reversible characteristics of phytochrome-mediated processes. By experimenting with inhibitors of photosynthesis we could demonstrate that the light-triggered potential changes in green bean hooks are correlated to photosynthetic electron transport phenomena. The red-light-induced transient is a depolarization, whereas blue light induces a hyperpolarization. Etiolated beans exhibit no bioelectric potential changes when subjected to red or far red irradiations. Blue light and white light induce a strong hyperpolarization in etiolated hooks cells. This transient seems to be an action potential induced by light. The action potential is influenced by inhibitors of electron transport and oxidative phosphorylation. By comparing the action spectrum of the action potential induced by light with the absorption spectra of extracted carotenoids and xanthophylls from etiolated bean hypocotyl hooks, we observed similarities.     

Assay of substances stimulatory to legume nodule formation

Two methods were developed for the assay of substances stimulatory to the nodulation of bean (Phaseolus vulgaris) roots growing from segments of hypocotyl tissue. Coconut water was the chief source of active material, but extracts of cotyledons, hypocotyls and leaves of beans and of horse chestnut fruits were also stimulatory. High concentrations of nitrate improved nodulation both in the presence and absence of coconut water. The ash of coconut water was inactive. Whole alfalfa seedlings formed nodules in the dark when grown in the split medium, but nodulation was not improved by the addition of coconut water.     

Auxin and red light in the control of hypocotyl hook opening in beans

Evidence is presented to support the suggestion that endogenous auxinlike substances participate in controlling the unbending of the hypocotyl hook of Phaseolus vulgaris L. (cv. Black Valentine). An acidic indole was detected in hook diffusates by fluorometry; triiodobenzoic acid, an inhibitor of auxin transport, prevented red light-induced unbending, and indoleacetic acid can be substituted for tissue just above the elbow region as an inhibitor of opening. Indoleacetic acid also stimulated growth of shank cells, and red light increased the sensitivity of this tissue to the hormone. A small red light-induced stimulation of auxin transport through the inside half of the hypocotyl shank was observed and may be related to light-induced unbending of the hook.     

Characteristics of hook formation by bean seedlings

Explants were isolated from 6-day-old etiolated bean seedlings (Phaseolus vulgaris L. cv. Black Valentine) containing the cotyledons with 4 mm of hypocotyl just below the node and/or the epicotyl. During incubation on distilled water, uneven growth of the hypocotyl or epicotyl occurred resulting in the formation of a hook. The more rapid growth of the side which became convex was not dependent upon the presence of the slower growing concave side. It was concluded that the main axis has an intrinsic capacity for asymmetric growth. The growth leading to hook formation was inhibited by α-naphthaleneacetic acid at concentrations above 0.2 milligram per liter.     

Acetylcholine and Synthesis of Ethylene in Etiolated Bean Tissues

Acetylcholine chloride inhibited ethylene production in etiolated bean tissues and prevented to some extent the IAA-promoted opening of the bean hypocotyl hooks. The results were interpreted on the basis of acetylcholine mimicking the effects of red light on auxin content and ethylene production.