Regulation of differential growth in the apical hook of Arabidopsis.

Arabidopsis seedlings develop a hook-like structure at the apical part of the hypocotyl when grown in darkness. Differential cell growth processes result in the curved hypocotyl hook. Time-dependent analyses of the hypocotyl showed that the apical hook is formed during an early phase of seedling growth and is maintained in a sequential phase by a distinct process. Based on developmental genetic analyses of hook-affected mutants, we show that the hookless mutants (hls1, cop2) are involved in an early aspect of hook development. From time-dependent analyses of ethylene-insensitive mutants, later steps in hook maintenance were found to be ethylene sensitive. Regulation of differential growth was further studied through examination of the spatial pattern of expression of two hormone-regulated genes: an ethylene biosynthetic enzyme and the ethylene receptor ETR1. Accumulation of mRNA for AtACO2, a novel ACC (1-aminocyclopropane-1-carboxylic acid) oxidase gene, occurred within cells predominantly located on the outer-side of the hook and was tightly correlated with ethylene-induced exaggeration in the curvature of the hook. ETR1 expression in the apical hook, however, was reduced by ethylene treatment. Based on the expression pattern of ETR1 and AtACO2 in the hook-affected mutants, a model for hook development and maintenance is proposed.     

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.     

Exploiting the triple response of Arabidopsis to identify ethylene-related mutants.

Alterations in the response of dark-grown seedlings to ethylene (the "triple response") were used to isolate a collection of ethylene-related mutants in Arabidopsis thaliana. Mutants displaying a constitutive response (eto1) were found to produce at least 40 times more ethylene than the wild type. The morphological defects in etiolated eto1-1 seedlings reverted to wild type under conditions in which ethylene biosynthesis or ethylene action were inhibited. Mutants that failed to display the apical hook in the absence of ethylene (his1) exhibited reduced ethylene production. In the presence of exogenous ethylene, hypocotyl and root of etiolated his1-1 seedlings were inhibited in elongation but no apical hook was observed. Mutants that were insensitive to ethylene (ein1 and ein2) produced increased amounts of ethylene, displayed hormone insensitivity in both hypocotyl and root responses, and showed an apical hook. Each of the "triple response" mutants has an effect on the shape of the seedling and on the production of the hormone. These mutants should prove to be useful tools for dissecting the mode of ethylene action in plants.     

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.     

Light-Stimulated Apical Hook Opening in Wild-Type Arabidopsis thaliana Seedlings

Apical hook opening and cotyledon unfolding are characteristic responses that occur during deetiolation of dicotyledonous seedlings. Light-stimulated apical hook opening and cotyledon unfolding in etiolated Arabidopsis thaliana seedlings appears to involve the activities of multiple photosensory systems. Red, far-red, and blue light are all effective in stimulating these responses in Arabidopsis. Stimulation of hook opening by red light and low fluence blue light is inductive, far-red reversible, and exhibits reciprocity, as is characteristic of many low fluence-dependent phytochrome-mediated responses. Far-red and high-fluence blue light appear to stimulate hook opening and cotyledon unfolding through high-irradiance-response systems during long-term light treatments. Although a phytochrome high-irradiance-response system presumably mediates the responses in far-red light, the responses to high-fluence blue light may be mediated by a blue light-specific photosensory system.     

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.     

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.     

Auxin Transport Is Required for Hypocotyl Elongation in Light-Grown but Not Dark-Grown Arabidopsis

Many auxin responses are dependent on redistribution and/or polar transport of indoleacetic acid. Polar transport of auxin can be inhibited through the application of phytotropins such as 1-naphthylphthalamic acid (NPA). When Arabidopsis thaliana seedlings were grown in the light on medium containing 1.0 μm NPA, hypocotyl and root elongation and gravitropism were strongly inhibited. When grown in darkness, however, NPA disrupted the gravity response but did not affect elongation. The extent of inhibition of hypocotyl elongation by NPA increased in a fluence-rate-dependent manner to a maximum of about 75% inhibition at 50 μmol m⁻² s⁻¹ of white light. Plants grown under continuous blue or far-red light showed NPA-induced hypocotyl inhibition similar to that of white-light-grown plants. Plants grown under continuous red light showed less NPA-induced inhibition. Analysis of photoreceptor mutants indicates the involvement of phytochrome and cryptochrome in mediating this NPA response. Hypocotyls of some auxin-resistant mutants had decreased sensitivity to NPA in the light, but etiolated seedlings of these mutants were similar in length to the wild type. These results indicate that light has a significant effect on NPA-induced inhibition in Arabidopsis, and suggest that auxin has a more important role in elongation responses in light-grown than in dark-grown seedlings.     

Ethylene-mediated enhancement of apical hook formation in etiolated Arabidopsis thaliana seedlings is gibberellin dependent

Dark-grown Arabidopsis seedlings develop an apical hook by differential elongation and division of hypocotyl cells. This allows the curved hypocotyl to gently drag the apex, which is protected by the cotyledons, upwards through the soil. Several plant hormones are known to be involved in hook development, including ethylene, which causes exaggeration of the hook. We show that gibberellins (GAs) are also involved in this process. Inhibition of GA biosynthesis with paclobutrazol (PAC) prevented hook formation in wild-type (WT) seedlings and in constitutive ethylene response (ctr)1-1, a mutant that exhibits a constitutive ethylene response. In addition, a GA-deficient mutant (ga1-3) did not form an apical hook in the presence of the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC). Analysis of transgenic Arabidopsis seedlings expressing a green fluorescent protein (GFP)-repressor of ga1-3 (RGA) fusion protein suggested that ACC inhibits cell elongation in the apical hook by inhibition of GA signaling. A decreased feedback of GA possibly causes an induction of GA biosynthesis based upon the expression of genes encoding copalyl diphosphate synthase (CPS; GA1) and GA 2-oxidase (AtGA2ox1). Furthermore, expression of GASA1, a GA-response gene, suggests that differential cell elongation in the apical hook might be a result of differential GA-sensitivity.     

Cytokinin, acting through ethylene, restores gravitropism to Arabidopsis seedlings grown under red light.

Cytokinin replaces light in several aspects of the photomorphogenesis of dicot seedlings. Arabidopsis thaliana seedlings grown under red light have been shown to become disoriented, losing the negative hypocotyl gravitropism that has been observed in seedlings grown in darkness or white light. We report here that cytokinin at micromolar concentrations restores gravitropism to seedlings grown under red light. Cytokinin cancels the effect of red light on the gravity-sensing system and at the same time replaces light in the inhibition of hypocotyl elongation. Furthermore, application of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid acts similarly to cytokinin. Cytokinin cannot restore gravitropism under red light to an ethylene-insensitive mutant that is defective at the EIN2 locus. Stimulation of ethylene production, therefore, can explain the action of cytokinin in restoring negative gravitropism to the hypocotyls of Arabidopsis seedlings grown under continuous red light.     

Cytokinin-induced hypocotyl elongation in light-grown<Emphasis Type="Italic"> Arabidopsis</Emphasis> plants with inhibited ethylene action or indole-3-acetic acid transport

Cytokinins inhibit hypocotyl elongation in darkness but have no obvious effect on hypocotyl length in the light. However, we found that cytokinins do promote hypocotyl elongation in the light when ethylene action is blocked. A 50% increase in Arabidopsis thaliana (L.) Heynh. hypocotyl length was observed in response to N⁶-benzyladenine (BA) treatment in the presence of Ag⁺. The level of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid was strongly increased, indicating that ethylene biosynthesis was up-regulated by treatment with cytokinin. Furthermore, the effects of cytokinins on hypocotyl elongation were also tested using a series of mutants in the cascade of the ethylene-signal pathway. In the ethylene-insensitive mutants etr1-3 and ein2-1, cytokinin treatment resulted in hypocotyl lengths comparable to those of wild-type seedlings treated with both Ag⁺ and BA. A similar phenotypical response to cytokinin was observed when auxin transport was blocked by -naphthylphthalamic acid (NPA). Applied cytokinin largely restored cell elongation in the basal and middle parts of the hypocotyls of NPA-treated seedlings and at the same time abolished the NPA-induced decrease in indole-3-acetic acid levels. Our data support the hypothesis that, in the light, cytokinins interact with the ethylene-signalling pathway and conditionally up-regulate ethylene and auxin synthesis.     

Two dominant photomorphogenic mutations of Arabidopsis thaliana identified as suppressor mutations of hy2

By screening suppressor mutants of the hy2 mutation of Arabidopsis thaliana , two dominant photomorphogenic mutants, shy1-1D and shy2-1D , for two genetic loci designated as SHY1 and SHY2 ( s uppressor of hy 2 mutation) have been isolated. Both of these non-allelic, extragenic suppressor mutations of hy2 are located on chromosome 1 of the Arabidopsis genome. Both mutations suppress the elongated hypocotyl phenotype of hy2 by light-independent inhibition of hypocotyl growth as well as by increasing the effectiveness of light inhibition of hypocotyl elongation. The shy1-1D mutation is partially photomorphogenic in darkness with apical hook opening and reduced hypocotyl elongation. The shy2-1D mutant displays highly photomorphogenic characteristics in darkness such as true leaf development, cotyledon expansion, and extremely reduced hypocotyl growth. In regard to hypocotyl elongation, however, the shy2-1D mutation is still light sensitive. Examination of red/far-red light responses shows that the shy1-1D mutation suppresses the hypocotyl elongation of the hy2 mutation effectively in red light but not effectively in far-red light. The shy2-1D suppresses hypocotyl elongation of the hy2 mutation effectively in both red and far-red light. Both mutations can also suppress the early-flowering phenotype of hy2 and have a distinct pleiotropic effect on leaf development such as upward leaf rolling. The data obtained suggest that SHY1 and SHY2 represent a novel class of components involved in the photomorphogenic pathways of Arabidopsis . This is the first report on the identification of dominant mutations in the light signal transduction pathway of plants.     

Photomorphogenic mutants of Arabidopsis thaliana reveal activities of multiple photosensory systems during light-stimulated apical-hook opening

Fluence rate-response curves were generated for red-, far-red-, and blue-light-stimulated apical-hook opening in seedlings of several photomorphogenic mutants of Arabidopsis thaliana (L.) Heynh. Compared to wild-type plants, hook opening was reduced in the phytochrome-deficient hy1, hy2, and hy6 mutants in red and far-red light at all fluence rates tested, and in low-fluence blue light, but was normal under high-irradiance blue light. In contrast, the blue-light-response mutants (blu1, blu2, and blu3) lacked the high-irradiance-dependent hook-opening response in blue light while hook opening was normal in low-fluence blue light and in red and farred light at all fluence rates tested. Hook opening in the phytochrome-B-deficient hy3 mutant was similar to wild type in all light conditions tested. The effects of the different mutations on light-induced hook opening indicate that a phytochrome(s) other than phytochrome B mediates hook opening stimulated by red, far-red and lowfluence blue light, while a blue-light-absorbing photoreceptor mediates the blue-light-sensitive high-irradiance response. Although the phytochrome and blue-light photosensory systems appear to work independently for the most part, some of their signal-transduction components may interact since the hy4, and hy5 mutants showed reduced hook-opening responses under conditions dependent on the phytochrome and blue-light-photosensory systems.We thank Jeff Young and Brian Parks for their many helpful suggestions during the progress of this research. This work was supported by National Science Foundation Grant No. DCB-9106697.     

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 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.     

Arabidopsis Mutants Lacking Blue Light-Dependent Inhibition of Hypocotyl Elongation

We have isolated a new class of photomorphogenic mutants in Arabidopsis. Hypocotyl elongation is not inhibited in the mutant seedlings by continuous blue light but is inhibited by far red light, indicating that these mutations are phenotypically different from the previously isolated long hypocotyl (hy) mutants. Complementation analysis indicated that recessive nuclear mutations at three genetic loci, designated blu1, blu2, and blu3, can result in the blu mutant phenotype and that these mutants are genetically distinct from other long hypocotyl mutants. The BLU genes appear to be important only during seedling development because the blu mutations have little effect on mature plants, whereas hypocotyl elongation and cotyledon expansion are altered in seedlings. The genetic separation of the blue and far red sensitivities of light-induced hypocotyl inhibition in the blu and hy mutants demonstrates that two photosensory systems function in this response.     

Phytochrome A regulates the intracellular distribution of phototropin 1-green fluorescent protein in Arabidopsis thaliana

It has been known for decades that red light pretreatment has complex effects on subsequent phototropic sensitivity of etiolated seedlings. Here, we demonstrate that brief pulses of red light given 2 h prior to phototropic induction by low fluence rates of blue light prevent the blue light-induced loss of green fluorescent protein-tagged phototropin 1 (PHOT1-GFP) from the plasma membrane of cortical cells of transgenic seedlings of Arabidopsis thaliana expressing PHOT1-GFP in a phot1-5 null mutant background. This red light effect is mediated by phytochrome A and requires approximately 2 h in the dark at room temperature to go to completion. It is fully far red reversible and shows escape from photoreversibility following 30 min of subsequent darkness. Red light-induced inhibition of blue light-inducible changes in the subcellular distribution of PHOT1-GFP is only observed in rapidly elongating regions of the hypocotyl. It is absent in hook tissues and in mature cells below the elongation zone. We hypothesize that red light-induced retention of the PHOT1-GFP on the plasma membrane may account for the red light-induced increase in phototropic sensitivity to low fluence rates of blue light.     

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.