Arabidopsis Hypocotyl Research Articles

Multiple photoreceptors mediate the light-induced reduction of GUS-COP1 from Arabidopsis hypocotyl nuclei.

The subcellular localization of COP1, a key photomorphogenic repressor, is regulated by light in Arabidopsis seedlings. Photoreceptor loss-of-function mutants and dominant gain-of-function overexpression transgenes were both used to analyze the influences of the three photoreceptors, phyA, phyB, and CRY1, on the light-regulated subcellular localization of COP1. Through a semiquantitative analysis of the nuclear abundance of GUS-COP1 in the various genetic backgrounds, the specific roles of the individual photoreceptors have been established. The data suggest that multiple photoreceptors influence the light-regulated subcellular localization of COP1 in white light. Under specific wavelengths of light, phyA, phyB, and CRY1 each play critical roles in mediating far-red, red, and blue light signals, respectively. Our data also support an interdependency between CRY1 and the phytochromes in mediating the light-regulated subcellular localization of COP1 and thus seedling development.

Interaction of cryptochrome 1, phytochrome, and ion fluxes in blue-light-induced shrinking of Arabidopsis hypocotyl protoplasts.

Protoplasts isolated from red-light-adapted Arabidopsis hypocotyls and incubated under red light exhibited rapid and transient shrinking within a period of 20 min in response to a blue-light pulse and following the onset of continuous blue light. Long-persisting shrinkage was also observed during continuous stimulation. Protoplasts from a hy4 mutant and the phytochrome-deficient phyA/phyB double mutant of Arabidopsis showed little response, whereas those from phyA and phyB mutants showed a partial response. It is concluded that the shrinking response itself is mediated by the HY4 gene product, cryptochrome 1, whereas the blue-light responsiveness is strictly controlled by phytochromes A and B, with a greater contribution by phytochrome B. It is shown further that the far-red-absorbing form of phytochrome (Pfr) was not required during or after, but was required before blue-light perception. Furthermore, a component that directly determines the blue-light responsiveness was generated by Pfr after a lag of 15 min over a 15-min period and decayed with similar kinetics after removal of Pfr by far-red light. The anion-channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid prevented the shrinking response. This result, together with those in the literature and the kinetic features of shrinking, suggests that anion channels are activated first, and outward-rectifying cation channels are subsequently activated, resulting in continued net effluxes of Cl- and K+. The postshrinking volume recovery is achieved by K+ and Cl- influxes, with contribution by the proton motive force. External Ca2+ has no role in shrinking and the recovery. The gradual swelling of protoplasts that prevails under background red light is shown to be a phytochrome-mediated response in which phytochrome A contributes more than phytochrome B.

Stomata Patterning on the Hypocotyl ofArabidopsis thalianaIs Controlled by Genes Involved in the Control of Root Epidermis Patterning

Stomata complexes are epidermal specialized structures typical of the upper aerial part of plants (shoot). In the model plantArabidopsis thaliana,we show that in the hypocotyl (the junction between the shoot and the root), stomata are organized according to a clear pattern reminiscent of the root epidermis pattern. Although stomata complexes are typical of the shoot epidermis, their pattern on the hypocotyl is under the control of genes involved in root epidermis patterning. Moreover, we have isolated a GFP marker line for the hypocotyl epidermal cells which do not differentiate stomata complexes. In this line the root and the hypocotyl epidermal patterns are similar. Our data support the existence of interactions between developmental mechanisms involved in the control of the apical/basal polarity and the radial symmetry of the plant body.

Phytochrome controls the number of endoreduplication cycles in the Arabidopsis thaliana hypocotyl.

A majority of the cells in the Arabidopsis hypocotyl undergo endoreduplication. The number of endocycles in this organ is partially controlled by light. Up to two cycles occur in light-grown hypocotyls, whereas in the dark about 30% of the cells go through a third cycle. Is the inhibition of the third endocycle in the light an indirect result of the reduced cell size in the light-grown hypocotyl, or is it under independent light control? To address this question, the authors examined the temporal and spacial patterns of endoreduplication in light- or dark-grown plants and report here on the following observations: (i) during germination two endocycles take place prior to any significant cell expansion; (ii) in the dark the third cycle is completed very early during cell growth; and (iii) a mutation that dramatically reduces cell size does not interfere with the third endocycle. The authors then used mutants to study the way light controls the third endocycle and found that the third endocycle is completely suppressed in far red light through the action of phytochrome A and, to a lesser extent, in red light by phytochrome B. Furthermore, no 16C nuclei were observed in dark-grown constitutive photomorphogenic 1 seedlings. And, finally the hypocotyl of the cryptochrome mutant, hy4, grown in blue light was about three times longer than that of the wild-type without a significant difference in ploidy levels. Together, the results support the view that the inhibition of the third endocycle in light-grown hypocotyls is not the consequence of a simple feed-back mechanism coupling the number of cycles to the cell volume, but an integral part of the phytochrome-controlled photomorphogenic program.

Anion-channel blockers interfere with auxin responses in dark-grown Arabidopsis hypocotyls.

Anion channels are thought to participate in signal transduction and turgor regulation in higher plant cells. The regulation of hypocotyl cell elongation is a situation in which these channels could play important roles because it involves ionic fluxes that are implicated in turgor control and orchestrated by various signals. We have used a pharmacological approach to reveal the contribution of anion channels in the regulation of the development of hypocotyls by auxins. Auxins induce an inhibition of elongation, a disintegration of the cortical cell layers, and the formation of adventitious roots on Arabidopsis thaliana hypocotyls grown in the dark. Anion-channel blockers such as anthracene-9-carboxylic acid, 4,4'-diisothiocyanatostilbene-2-2'-disulfonic acid, 4-acetamido-4'-isothiocyanato-stilbene-2-2'-disulfonic acid, and R(+)-methylindazone; indanyloxyacteic acid-94, which produce little or no stimulation of hypocotyl elongation by themselves, are able to counteract the inhibition and the disintegration induced by auxins with various efficiencies. This interference appears to be specific for auxins and does not occur when hypocotyl elongation is inhibited by other growth regulators such as ethylene or cytokinins. The putative involvement of anion channels in auxin signal transduction is discussed.

Voltage-dependent anion channel of Arabidopsis hypocotyls: nucleotide regulation and pharmacological properties.

Plasma membrane anion channels are thought to play important roles in osmoregulation and signal transduction in higher plant cells. Knowledge of their pharmacology and regulation is of importance to unravel their physiological functions. In this study, we explore the pharmacological properties and the nucleotide regulation of the voltage-dependent anion channel of Arabidopsis hypocotyls. The pharmacological profile of this channel is characterized by a low sensitivity to most anion channel blockers. It is inhibited by niflumic acid with an IC50 of 80 microM, but poorly sensitive to IAA-94 and NPPB and insensitive to 9-AC and DIDS. Nucleotides alter the amplitude, the kinetics and the voltage-dependence of the channel. The main effect of nucleotides is a shift of the voltage-dependent gate of the channel toward depolarized potentials leading to a strong reduction of the current amplitude. This regulation does not require ATP hydrolysis as nonhydrolyzable ATP analogues-AMPPNP and ATP gamma S-also regulate the anion current. This suggests that a nucleotide binding site is involved in the regulation. The study of the properties of this putative nucleotide binding site reveals that (i) ATP regulates the channel with an EC50 of 0.7 mM, (ii) adenyl nucleotides modulate the channel with the following order of effectiveness: ATP > ADP > > AMP, and (iii) thiophosphate nucleotide analogues are the most potent agonists with EC50 in the range of 80 microM. The hypothesis that this regulation may couple the electrical properties of the membrane with the metabolic status of the cell is discussed.

Ethylene can stimulate Arabidopsis hypocotyl elongation in the light.

Ethylene inhibits hypocotyl elongation in etiolated Arabidopsis seedlings. However, when Arabidopsis was grown in the light in the presence of ethylene or its precursor 1-aminocyclopropane-1-carboxylic acid (ACC), a marked induction of hypocotyl elongation occurred. This resulted from an increase in cell expansion rather than cell division. The effects of ethylene and ACC were antagonized by the ethylene action inhibitor Ag+. The elongation response was absent or weakened in a set of ethylene-insensitive mutants (etr1-3, ein2-1, ein3-1, ein4, ain1-10, ein7). With the exception of ein4, the degree of inhibition of hypocotyl elongation was correlated with the strength of the ethylene-insensitive phenotype based on the triple response assay. In addition, the constitutive ethylene response mutant ctr1-1, grown in the light, had a longer hypocotyl than the wild type. Exogenous auxin also induced hypocotyl elongation in light-grown Arabidopsis. Again, the response was abolished by treatment with Ag+, suggesting that ethylene might be a mediator. The results showed that, depending on light conditions, ethylene can induce opposite effects on cell expansion in Arabidopsis hypocotyls.

An anion channel in Arabidopsis hypocotyls activated by blue light.

A rapid, transient depolarization of the plasma membrane in seedling stems is one of the earliest effects of blue light detected in plants. It appears to play a role in transducing blue light into inhibition of hypocotyl (stem) elongation, and perhaps other responses. The possibility that activation of a Cl- conductance is part of the depolarization mechanism was raised previously and addressed here. By patch clamping hypocotyl cells isolated from dark-grown (etiolated) Arabidopsis seedlings, blue light was found to activate an anion channel residing at the plasma membrane. An anion-channel blocker commonly known as NPPB 15-nitro-2-(3-phenylpropylamino)-benzoic acid] potently and reversibly blocked this anion channel. NPPB also blocked the blue-light-induced depolarization in vivo and decreased the inhibitory effect of blue light on hypocotyl elongation. These results indicate that activation of this anion channel plays a role in transducing blue light into growth inhibition.

Hooked on hormonesLehman, A., Black, R. and Ecker, J.R. (1996) HOOKLESS1, an ethylene response gene, is required for differential cell elongation in the Arabidopsis hypocotyl, Cell 85, 183–194

Hooked on hormonesLehman, A., Black, R. and Ecker, J.R. (1996) HOOKLESS1, an ethylene response gene, is required for differential cell elongation in the Arabidopsis hypocotyl, Cell 85, 183–194

HOOKLESS1, an Ethylene Response Gene, Is Required for Differential Cell Elongation in the Arabidopsis Hypocotyl

Bending in plant tissues results from differential cell elongation. We have characterized Arabidopsis “hookless” mutants that are defective in differential growth in the hypocotyl. HOOKLESS1 was cloned and its predicted protein shows similarity to a diverse group of N-acetyltransferases. HOOKLESS1 mRNA is increased by treatment with ethylene and decreased in the ethylene-insensitive mutant ein2. High level expression of HOOKLESS1 mRNA results in constitutive hook curvature. The morphology of the hookless hypocotyl is phenocopied by inhibitors of auxin transport or by high levels of endogenous or exogenous auxin. Spatial patterns of expression of two immediate early auxin-responsive genes are altered in hookless1mutants, suggesting that the ethylene response gene HOOKLESS1controls differential cell growth by regulating auxin activity.

Genetic and transgenic evidence that phytochromes A and B act to modulate the gravitropic orientation of Arabidopsis thaliana hypocotyls.

Hypocotyls of Arabidopsis thaliana exhibit negative gravitropism in the dark, growing against the gravity vector. The direction of growth is randomized in red light (R). In single mutants lacking either phytochrome A or B randomization of hypocotyl orientation in R is retained. However, a double mutant lacks this response, indicating that either phytochrome A or B is capable of inducing randomization and phytochrome A and B are the only phytochromes involved in this process. The induction of randomization was confirmed using lines that express to different levels PHYA and PHYB cDNAs. Overexpression of PHYA cDNAs induced randomization of hypocotyl orientation in the dark. Dark randomization was also seen in the phyB-1 mutant but not in two other phyB alleles, suggesting that dark randomization in the phyB-1 line may be due to a second mutation. When germination was induced by gibberellin, rather than exposure to brief white light, randomization in the dark associated with phytochrome A overproduction was not observed but was retained in the phyB-1 mutant. Overexpression of PHYB cDNAs induced a light-dependent randomization of hypocotyl orientation that responded to R:far-red light ratio. We conclude that the default situation in Arabidopsis hypocotyls is, therefore, negative gravitropism, and either phytochrome A or phytochrome B can mediate randomization.

ATP-Dependent Regulation of an Anion Channel at the Plasma Membrane of Protoplasts from Epidermal Cells of Arabidopsis Hypocotyls.

Although Arabidopsis is the object of many genetic and molecular biology investigations, relatively few studies deal with regulation of its transmembrane ion exchanges. To clarify the role of ion transport in plant development, organ-and tissue-specific ion channels must be studied. We identified a voltage-dependent anion channel in epidermal cells of Arabidopsis hypocotyls, thus providing a new example of the occurrence of voltage-dependent anion channels in a specific plant cell type distinct from the stomatal guard cell. The Arabidopsis hypocotyl anion channel is able to function under two modes characterized by different voltage dependences and different kinetic behaviors. This switch between a fast and a slow mode is controlled by ATP. In the presence of intracellular ATP (fast mode), the channels are closed at resting potentials, and whole-cell currents activate upon depolarization. After activation, the anion current deactivates rapidly and more and more completely at potentials negative to the peak. In the absence of ATP, the current switches from this fast mode to a mode characterized by a slow and incomplete deactivation at resting potentials. In addition, the whole-cell currents can be correlated with the activity of single channels. In the outside-out configuration, the presence of ATP modulates the mean lifetimes of the open and closed states of the channel at hyperpolarized potentials, thus controlling its open probability. The fact that ATP-dependent voltage regulation was observed in both whole-cell and outside-out configurations suggests that a single type of anion channel can switch between two modes with distinct functional properties.

ATP-Dependent Regulation of an Anion Channel at the Plasma Membrane of Protoplasts from Epidermal Cells of Arabidopsis Hypocotyls

ATP-Dependent Regulation of an Anion Channel at the Plasma Membrane of Protoplasts from Epidermal Cells of Arabidopsis Hypocotyls

Light inactivation of arabidopsis photomorphogenic repressor COP1 involves a cell-specific regulation of its nucleocytoplasmic partitioning

Arabidopsis COP1 acts as a repressor of photomorphogenesis in darkness, and light stimuli abrogate this suppressive action. COP1, when fused to β-glucuronidase (GUS), is enriched in the nucleus in darkness, but not in the light, in hypocotyl cells of Arabidopsis seedlings and epidermal cells of onion bulbs. In Arabidopsis hypocotyl cells, the nuclear GUS-COP1 level changes in response to dark-light transitions and quantitatively correlates with the extent of repression of photomorphogenic development. In root cells, GUS-COP1 is constitutively nuclear, consistent with an established role of COP1 in suppressing root chloroplast development in both light and darkness. We conclude that COP1 acts inside the nucleus to suppress photomorphogenesis and that light inactivation of COP1 involves a cell type-specific control of its nucleocytoplasmic partitioning.

Embryonic origin of the Arabidopsis primary root and root meristem initials

ABSTRACT The embryonic origin of the Arabidopsis root and hypocotyl region has been investigated using histological techniques and clonal analysis. Our data reveal the pattern of cell division in the embryo giving rise to the various initials within the root promeristem. A small region of the root at its connection with the hypocotyl appears not to be derived from the promeristem initials. This region contains two cortical cell layer and [3H]thymidine incorporation data suggest that it lacks postembryonic cell divisions. Sectors marked by transposon excision from the β-glucuronidase marker gene are used to investigate cell lineages giving rise to root and hypocotyl. The position of end points from sectors with embryonic origin show little variation and hence reveal preferred positions in the seedling for cells derived from different regions of the embryo. The radial extent of complete root sectors is consistent with the radial arrangement of root meristem initials at the heart stage of embryogenesis inferred from histological analysis. Using the clonal data, a fate map is constructed depicting the destiny of heart stage embryonic cell tiers, in the seedling root and hypocotyl. The variability in the sector end points indicates that distinct cell lineages are not restricted for root or hypocotyl fate. In contrast, derivatives of the hypophyseal cell do appear to be restricted to the columella and central cell region of the root.