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 (Arabidopsis thaliana). We used a peptide of the carboxyl-terminal region of AtABP1 as a tool. We performed mutant analysis with the null alleles of ABP1, abp1-c1 and abp1-TD1, and the TILLING mutant abp1-5 We employed Coimbra, an accession that exhibits an amino acid exchange in the auxin-binding domain of ABP1. We measured either volume changes of single hypocotyl protoplasts or hypocotyl growth, both at high temporal resolution. 5-F-IAA selectively activated the TIR1/AFB pathway but did not induce protoplast swelling; instead, it showed auxin activity in the hypocotyl growth test. In contrast, PEO-IAA induced an auxin-like swelling response but no hypocotyl growth. The carboxyl-terminal peptide of AtABP1 induced an auxin-like swelling response. In the ABP1-related mutants and Coimbra, no auxin-induced protoplast swelling occurred. ABP1 seems to be involved in mediating rapid auxin-induced protoplast swelling, but it is not involved in the control of rapid auxin-induced growth.

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 tremendous amount of digital image data are automatically analysed using techniques of 'machine vision'. This minireview will shed light on the available imaging strategies and discuss standard methods for the automated analysis of images to give the non-bioinformatic reader an idea how the new technology works. A number of successful platforms will be described and the prospects that image-based phenomics may offer for elucidating hormonal cross-talk and molecular growth physiology will be discussed.

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. We developed Hansa Trace, software for automatically detecting and measuring hypocotyl segments and roots in the images. We validated this system by measuring some well-characterized growth responses to auxins, non-auxins, ATPase activators and apoplastic acidification. The method can be shared on a cooperation basis and is able to perform measurements with minimal user intervention.

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. In contrast with dwarfed twd1 loss-of-function alleles, TWD1 gain-of-function lines that lack a putative in-plane membrane anchor (HA-TWD1-Ct ) show hypermorphic plant architecture, characterized by enhanced stem length and leaf surface but reduced shoot branching. Greater hypocotyl length is the result of enhanced cell elongation that correlates with reduced polar auxin transport capacity for HA-TWD1-Ct . As a consequence, HA-TWD1-Ct displays higher hypocotyl auxin accumulation, which is shown to result in elevated auxin-induced cell elongation rates. Our data highlight the importance of C-terminal membrane anchoring for TWD1 action, which is required for specific regulation of ABCB-mediated auxin transport. These data support a model in which TWD1 controls lateral ABCB1-mediated export into the apoplast, which is required for auxin-mediated cell elongation.

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. We measured the IAA-induced growth inhibition at high time resolution and show that the F-box auxin receptor mutants failed to display this effect. We conclude that the F-box auxin receptors are needed for the response. In hypocotyls, auxin induces an increase in elongation growth, and this effect has been earlier shown to be independent of the F-box receptors. Based on these findings, we discuss differences in the growth control modes in roots and shoots. We demonstrate that the rapid auxin-induced root growth inhibition, unlike the induction of growth in hypocotyls, requires the presence 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). Antibodies raised against box a of ABP1 induce protoplast swelling in the absence of auxin. This response is inhibited by pre-incubation with 2-NAA. The effect of 2-NAA on swelling induced by agonistic antibodies appears to depend on the binding characteristics of the antibody. ScFv12, an antibody directed against box a, box c and the C-terminal domain of ABP1 also exhibits auxin-agonist activity which is, however, not abolished by 2-NAA. Neither does 2-NAA affect the activity of the C-terminal peptide of ABP1, which is predicted to interact with putative binding proteins of ABP1. These results support the view that box a and box c of ABP1 are auxin-binding domains.

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. It was shown that WH7 acts in a genetically defined auxin pathway. These small molecules will extend the arsenal of substances that can be used to define auxin perception site(s) and to dissect subsequent signalling events.

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. In a site-selected approach, we were able to identify three independent alleles of Mutator-transposon insertions in Zmk1. zmk1-m1::Mu1 plants were characterised by a Mu1 transposon inside intron 1 of ZMK1. When we analysed the Zmk1-transcript abundance in growing coleoptiles of these homozygous mutants, however, we found the K(+)-channel allele overexpressed. In consequence, elevated levels of K(+)-channel transcripts resulted in a growth phenotype as expected from more efficient K(+)-uptake, representing a central factor for turgor formation. Following Zmk1 expression during maize embryogenesis, we found this K(+)-channel gene constitutively expressed throughout embryo development and upregulated in late stages. In line with a vital role in embryogenesis, the mutations of exon 2 and intron 2 of Zmk1-zmk1-m2::Mu8 and zmk1-m3::MuA2-caused a lethal, defective-kernel phenotype. Thus, these results demonstrate the central role of the auxin-regulated K(+)-channel gene Zmk1 in coleoptile growth and embryo development.

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. Among the members of the Shaker-like K+ channels, we thereby identified transcripts of the inward rectifiers, KAT1 (K+ transporter of Arabidopsis thaliana) and KAT2, to be upregulated by auxin. The phloem-associated KAT2 was localised in cotyledons and the apical part of etiolated seedlings. In contrast, the K+-channel gene KAT1 was expressed in the cortex and epidermis of etiolated hypocotyls, as well as in flower stalks. Furthermore, KAT1 was induced by active auxins in auxin-sensitive tissues characterised by rapid cell elongation. Applying the patch-clamp technique to protoplasts of etiolated hypocotyls, we correlated the electrical properties of K+ currents with the expression profile of K+-channel genes. In KAT1-knockout mutants, K+ currents after auxin stimulation were characterised by reduced amplitudes. Thus, this change in the electrical properties of the K+-uptake channel in hypocotyl protoplasts resulted from an auxin-induced increase of active KAT1 proteins. The loss of KAT1-channel subunits, however, did not affect the auxin-induced growth rate of hypocotyls, pointing to compensation by residual, constitutive K+ transporters. From gene expression and electrophysiological data, we suggest that auxin regulation of KAT1 is involved in elongation growth of Arabidopsis. Furthermore, a role for KAT2 in the auxin-controlled vascular patterning of leaves is discussed.

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. Protoplasts isolated from wild-type hypocotyls respond to auxin by a rapid increase in cell volume, which we measured by image analysis at a high temporal resolution. A similar swelling could be triggered by antibodies directed against a part of the putative auxin-binding domain (box-a) of the auxin-binding protein 1 (ABP1). Induction of swelling both by auxin and by the antibody was not observed in the protoplasts isolated from the dgt mutant. However, dgt protoplasts are able to respond to the stimulator of the H(+)-ATPase, fusicoccin, with normal swelling. We propose that dgt is a signal-transduction mutation interfering with an auxin-signalling pathway that uses ABP1 as a receptor.

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. Cytokinin treatment also inhibited rapid auxin responses in hypocotyl segments: auxin-stimulated elongation, H(+) secretion, and ethylene synthesis were all inhibited by cytokinin in wild-type hypocotyl segments, and thus mimicked the impaired auxin responsiveness found in dgt hypocotyls. However, cytokinin failed to inhibit auxin-induced LeSAUR gene expression, an auxin response that is affected by the dgt mutation. In addition, cytokinin treatment inhibited the auxin induction of only one of two 1-aminocyclopropane-1-carboxylic acid synthase genes that exhibited impaired auxin inducibility in dgt hypocotyls. Thus, cytokinin inhibited a subset of the auxin responses impaired in dgt hypocotyls, suggesting that cytokinin blocks at least one branch of the DGT-dependent auxin response pathway.

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.). Abraded hypocotyl segments of 4-day-old, etiolated dgt seedlings showed an impaired H+ secretion response to applied auxin. mRNA levels for two PM H+-ATPase isoforms, LHA2 and LHA4, were not reduced in dgt hypocotyl segments as compared to wild-type segments, suggesting that the dgt mutation does not affect H+ secretion by reducing the transcription of major PM H+-ATPase genes. The dgt mutation also disrupted auxin inhibition of growth and H+ secretion in roots of 4-day-old dgt seedlings. However, immediately after germination, dgt seedling roots responded to auxin with a near-normal inhibition of growth. In addition, stomata in epidermal peels from 2-week-old dgt cotyledons demonstrated normal auxin-induced opening. We conclude that an intact DGT gene product is required for auxin-induced H+ secretion in tomato hypocotyl segments and for auxin inhibition of H+ secretion in roots of older seedlings, but that a DGT-independent pathway for auxin responses exists in young root tips and in guard cells. A developmentally controlled switch from DGT-independent to DGT-dependent auxin signalling appears to take place in root tips within 2 days after germination.