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.     

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.     

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.     

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.     

Hormone-solute interactions in the lettuce hypocotyl hook

The hypocotyl hook of the lettuce (Lactuca sativa cv. Grand Rapids) seedling is stimulated to a high degree of curvature through a synergistic interaction of ethylene and gibberellic acid in the light. Presentation of various inorganic salts to the seedlings caused extensive alteration of the hormone-induced curvatures, with ammonium and sulfate being the most stimulatory of curvature, and potassium and carbonate being the most inhibitory of curvature. Experiments using organic buffers indicated that the effect was not a pH response. The abilities of various cations and anions to alter the hormonally regulated curvature is suggested as further evidence of solute alteration of hormonal effectiveness. The interpretation is offered that the solutes may be influencing hormonal effectiveness through salting-in and salting-out effects on macro-molecules such as proteins.     

Auxin, ethylene and brassinosteroids: tripartite control of growth in the Arabidopsis hypocotyl

Dark-grown Arabidopsis seedlings develop an apical hook by differential cell elongation and division, a process driven by cross-talk between multiple hormones. Auxins, ethylene and gibberellins interact in the formation of the apical hook. In the light, a similar complexity of hormonal regulation has been revealed at the level of hypocotyl elongation. Here, we describe the involvement of brassinosteroids (BRs) in auxin- and ethylene-controlled processes in the hypocotyls of both light- and dark-grown seedlings. We show that BR biosynthesis is necessary for the formation of an exaggerated apical hook and that either application of BRs or disruption of BR synthesis alters auxin response, presumably by affecting auxin transport, eventually resulting in the disappearance of the apical hook. Furthermore, we demonstrate that ethylene-stimulated hypocotyl elongation in the light is largely controlled by the same mechanisms as those governing formation of the apical hook in darkness. However, in the light, BRs appear to compensate for the insensitivity to ethylene in hls mutants, supporting a downstream action of BRs. Hence, our results indicate that HLS1, SUR1/HLS3/RTY1/ALF1 and AMP1/HPT/COP2/HLS2/PT act on the auxin-ethylene interaction, rather than at the level of BRs. A model for the tripartite hormone interactions is presented.     

Auxin and gibberellin responsive Arabidopsis SMALL AUXIN UP RNA36 regulates hypocotyl elongation in the light

Key message

The Arabidopsis SAUR36 , renamed RAG1 , integrates auxin and gibberellin signals to regulate apical hook maintenance in etiolated seedlings, hypocotyl elongation in the light and fertility.

Abstract

Phytohormone signalling intermediates integrate responses to developmental cues and the variety of environmental inputs thereby governing all aspects of plant growth and development. At the genetic level, interactions of different phytohormone signalling pathways lead to the regulation of overlapping sets of target genes. We have characterised SMALL AUXIN UP RNA 36 (SAUR36, At2g45210) whose expression is induced by auxins and repressed by gibberellins. Its expression appears to be restricted to elongating tissues. Germination responses to treatments with paclobutrazol and exogenous abscisic acid were affected in knock-out, knock-down as well as ectopic expression lines. At later stages of development, however, transgenic plants with reduced levels of SAUR36 expression appeared similar to wild-type plants, while ectopic expression of SAUR36 led to the absence of apical hooks in etiolated seedlings and longer hypocotyls in light-grown seedlings. Mature plants ectopically expressing SAUR36 further displayed strongly reduced fertility and wavy growth of inflorescence axes, the latter of which could be linked to defects in auxin transport. Taken together, our data suggest that SAUR36 plays a role in the regulation of seed germination by gibberellins and abscisic acid, light-dependent hypocotyl elongation as well as apical hook formation or maintenance. Therefore, we propose that it could act as one of the converging points of auxin and gibberellin signal integration in controlling key plant developmental events. Hence, we named the gene RESPONSE TO AUXINS AND GIBBERELLINS 1 (RAG1).     

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.     

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.     

Photocontrol of the hypocotyl hook opening of Sinapis alba seedlings. Involvement of phytochrome and a high irradiance response

Baumgartner, N. and Fondeville, J. C. 1989. Photocontrol of the hypocotyl hook opening of Sinapis alba seedlings. Involvement of phytochrome and a high irradiance response.
A statistical evaluation of the hypocotyl hook opening (hook opening index) was used for measurement of the hook angle in lots of etiolated Sinapis alba L. cv. Albatros seedlings. Studies of the kinetics for hook opening were carried out in continuous fluorescent white, blue and red light (6, 15 and 40 μmol m^-2s^-1) with 2-day-old dark-grown seedlings. At the beginning of the irradiation period the photoresponse in red light was the opposite to that in blue (low photon fluences). Blue rapidly induced the hook opening (in less than 20 min), while red produced hook tightening (photon fluences up to 70 mmol m^-2), which precedes the normal progressive hook opening. For low fluences, the data were consistent with the involvement of phytochrome and a specific blue light photoreceptor. A phytochrome effect was observed in the hook opening, dependent upon a high irradiance response (HIR). This HIR (like that for the inhibition of the hypocotyl elongation) was characterized by a wavelength response curve with maxima in the blue and far-red regions of the spectrum.     

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.     

Auxin increases the hydraulic conductivity of auxin-sensitive hypocotyl tissue

The ability of water to enter the cells of growing hypocotyl tissue was determined in etiolated soybean (Glycine max (L.) Merr.) seedlings. Water uptake was restricted to that for cell enlargement, and the seedlings were kept intact insofar as possible. Tissue water potentials ( _w) were measured at thermodynamic equilibrium with an isopiestic thermocouple psychrometer. _wwas below the water potential of the environment by as much as 3.1 bars when the tissue was enlarging rapidly. However, _w was similar to the water potential of the environment when cell enlargement was not occurring. The low _w in enlarging tissue indicates that there was a low conductivity for water entering the cells.The ability of water to enter the enlarging cells was defined as the apparent hydraulic conductivity of the tissue (Lp). Despite the low Lp of growing cells, Lp decreased further as cell enlargement decreased when intact hypocotyl tissue was deprived of endogenous auxin (indole-3-acetic acid) by removal of the hypocotyl hook. Cell enlargement resumed and Lp increased when auxin was resupplied exogenously. The auxin-induced increase in Lp was correlated with the magnitude of the growth enhancement caused by auxin, and it was observed during the earliest phase of the growth response to auxin. The increase in Lp appeared to be caused by an increase in the hydraulic conductivity of the cell protoplasm, since other factors contributing to Lp remained constant. The rapidity of the response is consistent with a cellular site of action at the plasmalemma, although other sites are not precluded.Because the experiments involved only short times, auxin-induced changes in cell enlargement could not be attributed to changes in cell osmotic potentials. Neither could they be attributed to changes in turgor, which increased when the rate of enlargement decreased. Rather, auxin appeared to act by altering the extensibility of the cell walls and by simultaneously altering the ability of water to enter the growing cells under a given water potential gradient. The hydraulic conductivity and extensibility of the cell walls appeared to contribute about equally to the control of the growth rate of the hypocotyls.     

Influence of acetylcholine and light on the bioelectric potential of bean (Phaseolus vulgaris L.) hypocotyl hook

Acetylcholine did not show a short-time effect on the extracellularbioelectric potential differences of etiolated bean (Phaseolusvulgaris L.) hypocotyls. Acetylcholine in the dark did not mimicany light effect but influenced the photoelectric responsesinduced by blue light. Hyperpolarization was inhibited by increasingthe acetylcholine concentration. This effect seemed to dependon the increasing hyperpolarization of the resting potentialof the hooks during incubation with acetylcholine. Potassiumchloride showed the same effect on the photoelectric responsebut in this case, we found a more positive resting potentialwith increasing salt concentrations. The potassium content ofhypocotyl hooks incubated in the dark in 0.2 M KCl solutionwith acetylcholine was significantly less than in the controls. (Received May 31, 1977; )     

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.     

Dual effect of phytochrome A on hypocotyl growth under continuous red light

The role of phytochrome A in the control of hypocotyl growth under continuous red light (Rc) was investigated using phyA and phyB mutants of Arabidopsis thaliana, which lack phytochrome A (phyA) or phytochrome B (phyB), respectively, and transgenic seedlings of Nicotiana tabacum overexpressing Avena phyA, compared to the corresponding wild type (WT). In WT seedlings of A. thaliana, hypocotyl growth inhibition showed a biphasic response to the fluence rate of Rc, with a brake at 10^?2μmol m^?2 s^?1. At equal total fluence rate, hourly pulses of red light caused slightly more inhibition than Rc. The response to very low fluences of continuous or pulsed red light was absent in the phyA and phyA phyB mutants and present in the phyB mutant. The second part of the response was steeper in the phyA mutant than in the WT but was absent in the phyB mutant. In WT tobacco the response to Rc was biphasic. Overexpression of Avena phyA enhanced the response only at very low fluence rates of Rc (< 10^?2μmol m^?2 s^?1). In both species, the effect of hourly pulses of far-red light was similar to the maximum inhibition observed in the first phase of the response to Rc. Using reciprocity failure (i.e. higher inhibition under continuous than pulsed light) as the operational criterion, a ‘true’ high-irradiance reaction occurred under continuous far-red light but not under Rc or red plus far-red light mixtures. Native and overexpressed phyA are proposed to mediate very low fluence responses under Rc. In WT A. thaliana, this effect is counteracted by a negative action of phyA on phyB-mediated low-fluence responses.     

Auxin and light control of adventitious rooting in Arabidopsis require ARGONAUTE1

Adventitious rooting is a quantitative genetic trait regulated by both environmental and endogenous factors. To better understand the physiological and molecular basis of adventitious rooting, we took advantage of two classes of Arabidopsis thaliana mutants altered in adventitious root formation: the superroot mutants, which spontaneously make adventitious roots, and the argonaute1 (ago1) mutants, which unlike superroot are barely able to form adventitious roots. The defect in adventitious rooting observed in ago1 correlated with light hypersensitivity and the deregulation of auxin homeostasis specifically in the apical part of the seedlings. In particular, a clear reduction in endogenous levels of free indoleacetic acid (IAA) and IAA conjugates was shown. This was correlated with a downregulation of the expression of several auxin-inducible GH3 genes in the hypocotyl of the ago1-3 mutant. We also found that the Auxin Response Factor17 (ARF17) gene, a potential repressor of auxin-inducible genes, was overexpressed in ago1-3 hypocotyls. The characterization of an ARF17-overexpressing line showed that it produced fewer adventitious roots than the wild type and retained a lower expression of GH3 genes. Thus, we suggest that ARF17 negatively regulates adventitious root formation in ago1 mutants by repressing GH3 genes and therefore perturbing auxin homeostasis in a light-dependent manner. These results suggest that ARF17 could be a major regulator of adventitious rooting in Arabidopsis.     

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.     

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; )     

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.