Protoplast Swelling and Hypocotyl Growth Depend on Different Auxin Signaling Pathways.

Members of the TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX PROTEIN (TIR1/AFB) family are known auxin receptors. To analyze the possible receptor function of AUXIN BINDING PROTEIN1 (ABP1), an auxin receptor currently under debate, we performed different approaches. We performed a pharmacological approach using α-(2,4-dimethylphenylethyl-2-oxo)-indole-3-acetic acid (auxinole), α-(phenylethyl-2-oxo)-indole-3-acetic acid (PEO-IAA), and 5-fluoroindole-3-acetic acid (5-F-IAA) to discriminate between ABP1- and TIR1/AFB-mediated processes in Arabidopsis ().

Novel imaging-based phenotyping strategies for dissecting crosstalk in plant development.

In an era of genomics, proteomics, and metabolomics a large number of mutants are available. The discovery of their phenotypes is fast becoming the bottleneck of molecular plant physiology. This crisis can be overcome by imaging-based phenotyping, an emerging, rapidly developing and innovative approach integrating plant and computer science.

A high-throughput imaging auxanometer for roots and hypocotyls of Arabidopsis using a 2D skeletonizing algorithm.

Next generation phenotyping of auxin response mutants will be greatly facilitated by the ability to record rapid growth responses in roots and hypocotyls at high throughput and at high temporal resolution. As Arabidopsis seedlings are very tiny and fragile, imaging is the only adequate way for data acquisition. As camera-based systems described before have a limited throughput, we used commercial flatbed scanners to record a large number of simultaneous experiments.

Expression of TWISTED DWARF1 lacking its in-plane membrane anchor leads to increased cell elongation and hypermorphic growth.

Plant growth is achieved predominantly by cellular elongation, which is thought to be controlled on several levels by apoplastic auxin. Auxin export into the apoplast is achieved by plasma membrane efflux catalysts of the PIN-FORMED (PIN) and ATP-binding cassette protein subfamily B/phosphor-glycoprotein (ABCB/PGP) classes; the latter were shown to depend on interaction with the FKBP42, TWISTED DWARF1 (TWD1). Here by using a transgenic approach in combination with phenotypical, biochemical and cell biological analyses we demonstrate the importance of a putative C-terminal in-plane membrane anchor of TWD1 in the regulation of ABCB-mediated auxin transport.

Rapid auxin-induced root growth inhibition requires the TIR and AFB auxin receptors.

We investigated the relation between auxin-induced gene expression and the rapid auxin-induced growth inhibition in Arabidopsis thaliana roots. The natural auxin indole-3-acetic acid (IAA) induced a strong activation of gene expression as visualized by the DR5rev::GFP reporter gene technique. This effect was specific for active auxins and was abolished in knockout mutants of the F-box auxin receptors.

Exploring the link between auxin receptors, rapid cell elongation and organ tropisms.

Auxin receptor F-box proteins of the TIR1/AFB family are known to regulate auxin-induced gene expression. We could demonstrate that rapid auxin-induced hypocotyl elongation, the most classical auxin response, is only mildly affected in Arabidopsis plants in which most of the receptor genes have been knocked out, while gene expression is almost completely abolished. Here we test the same receptor mutant plants for their gravitropic and phototropic responsiveness, generally considered to base on auxin gradients across the hypocotyl.

The auxin-binding pocket of auxin-binding protein 1 comprises the highly conserved boxes a and c.

The auxin-binding protein 1 (ABP1) has already been proved to be an extracellular receptor of auxin in single cell systems. Protoplasts of maize coleoptiles respond to auxin with an increase in volume. The 2-naphthaleneacetic acid (2-NAA), an inactive auxin analog, acts as an anti-auxin in protoplast swelling, as it suppresses the effect of indole-3-acetic acid (IAA).

Cold-induced cytosolic free calcium ion concentration changes in wheat.

Relatively little is known about changes in the cytosolic free calcium ion concentration ([Ca(2+)](c)) in monocotyledonous plants. Therefore, we produced transgenic winter wheat lines stably expressing the calcium-sensitive photoprotein aequorin constitutively in the cytosol. [Ca(2+)](c) was detected in vivo by luminometry, and [Ca(2+)](c) elevations were imaged at video rate.

Identification of auxins by a chemical genomics approach.

Thirteen auxenic compounds were discovered in a screen of 10 000 compounds for auxin-like activity in Arabidopsis roots. One of the most potent substances was 2-(4-chloro-2-methylphenoxy)-N-(4-H-1,2,4-triazol-3-yl)acetamide (WH7) which shares similar structure to the known auxenic herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). A selected set of 20 analogues of WH7 was used to provide detailed information about the structure-activity relationship based on their efficacy at inhibiting and stimulating root and shoot growth, respectively, and at induction of gene expression.

The auxin-induced K(+) channel gene Zmk1 in maize functions in coleoptile growth and is required for embryo development.

The transcript level and in turn protein density of the K(+)-uptake channel ZMK1 in maize (Zea mays) coleoptiles is controlled by the phytohormone auxin. ZMK1 is involved in auxin-regulated coleoptile elongation as well as gravi- and phototropism. To provide unequivocal evidence for the role of ZMK1 in these elementary processes we screened for maize plants containing a Mutator-tagged Zmk1 gene.

How does auxin enhance cell elongation? Roles of auxin-binding proteins and potassium channels in growth control.

Elongation growth and a several other phenomena in plant development are controlled by the plant hormone auxin. A number of recent discoveries shed light on one of the classical problems of plant physiology: the perception of the auxin signal. Two types of auxin receptors are currently known: the AFB/TIR family of F box proteins and ABP1.

Auxin activates KAT1 and KAT2, two K+-channel genes expressed in seedlings of Arabidopsis thaliana.

The transcript abundance of the K+-channel gene ZMK1 (Zea mays K+ channel 1) in maize coleoptiles is controlled by the phytohormone auxin. Thus, ZMK1 is thought to function in auxin-regulated coleoptile elongation, as well as during gravitropism and phototropism. To investigate related growth phenomena in the dicotyledonous plant Arabidopsis thaliana, we screened etiolated seedlings for auxin-induced K+-channel genes.

The diageotropica mutation of tomato disrupts a signalling chain using extracellular auxin binding protein 1 as a receptor.

The diageotropica ( dgt) mutant of tomato ( Lycopersicon esculentum Mill.) is known to lack a number of typical auxin responses. Here we show that rapid auxin-induced growth of seedling hypocotyls is completely abolished by the mutation over the full range of auxin concentrations tested, and also in early phases of the time course.

Cytokinin inhibits a subset of diageotropica-dependent primary auxin responses in tomato.

Many aspects of plant development are regulated by antagonistic interactions between the plant hormones auxin and cytokinin, but the molecular mechanisms of this interaction are not understood. To test whether cytokinin controls plant development through inhibiting an early step in the auxin response pathway, we compared the effects of cytokinin with those of the dgt (diageotropica) mutation, which is known to block rapid auxin reactions of tomato (Lycopersicon esculentum) hypocotyls. Long-term cytokinin treatment of wild-type seedlings phenocopied morphological traits of dgt plants such as stunting of root and shoot growth, reduced elongation of internodes, reduced apical dominance, and reduced leaf size and complexity.

Developmental regulation of H+-ATPase-dependent auxin responses in the diageotropica mutant of tomato (Lycopersicon esculentum).

Rapid auxin effects on H+ pumping across the plasma membrane precede auxin-induced elongation growth of hypocotyls and swelling of guard cells, as well as auxin inhibition of root growth. To investigate whether auxin-signalling mechanisms in such diverse cell types are similar, we characterized these responses in various tissues of the diageotropica (dgt) mutant of tomato (Lycopersicon esculentum Mill.).

The auxin signal for protoplast swelling is perceived by extracellular ABP1.

Protoplasts of corn coleoptiles and Arabidopsis hypocotyls respond to the plant hormone auxin with a rapid change in volume. We checked the effect of antibodies directed against epitopes of auxin-binding protein 1 from Arabidopsis thaliana (AtERabp1) and Zea mays (ZmERabp1), respectively. Antibodies raised against the C-terminus of AtERabp1 inhibited the response to auxin, while antibodies raised against a part of box a, the putative auxin-binding domain, induced a swelling response similar to that caused by auxin treatment.

Fusicoccin- and IAA-induced elongation growth share the same pattern of K+ dependence.

The dependence of growth induced by the fungal toxin fusicoccin (FC) on the K+ content of the incubation medium was investigated in abraded maize coleoptiles. If the divalent ion Ca2+ was included in the bathing medium, no FC-induced growth occurred in the absence of K+, whereas a strong response was detected in presence of K+. The optimal K+ concentration was in the range of 1-10 mM.

Auxin-induced K+ channel expression represents an essential step in coleoptile growth and gravitropism.

Auxin-induced growth of coleoptiles depends on the presence of potassium and is suppressed by K+ channel blockers. To evaluate the role of K+ channels in auxin-mediated growth, we isolated and functionally expressed ZMK1 and ZMK2 (Zea mays K+ channel 1 and 2), two potassium channels from maize coleoptiles. In growth experiments, the time course of auxin-induced expression of ZMK1 coincided with the kinetics of coleoptile elongation.

Oxidoreductases in plant plasma membranes.

Electron transporting oxidoreductases at biological membranes mediate several physiological processes. While such activities are well known and widely accepted as physiologically significant for other biological membranes, oxidoreductase activities found at the plasma membrane of plants are still being neglected. The ubiquity of the oxidoreductases in the plasma membrane suggests that the activity observed is of major importance in fact up to now no plant without redox activity at the plasmalemma is known.

Reexamination of the Acid growth theory of auxin action.

Some crucial arguments against the acid growth theory of auxin action (U Kutschera, P Schopfer [1985] Planta 163: 483-493) have been reinvestigated by simultaneous measurements of proton fluxes and growth of maize (Zea mays L.) coleoptiles. Special care was taken to obtain a mild, effective, and reproducible abrasion of the cuticle.

Hexachloroiridate IV as an Electron Acceptor for a Plasmalemma Redox System in Maize Roots.

Hexachloroiridate IV, a new artificial electron acceptor for the constitutive plant plasma membrane redox system has been investigated. It appeared not to permeate through biological membranes. Due to its higher redox potential, it is a more powerful electron acceptor than hexacyanoferrate III (ferricyanide) and even micromolar concentrations are rapidly reduced.